Methods and compositions for modulating fertility

ABSTRACT

Methods and compositions for inducing immune suppression are disclosed. The methods involve administering an effective amount of an OX-2 protein or a nucleic acid encoding an OX-2 protein. The methods are useful in preventing graft rejection, fetal loss, autoimmune disease, and allergies. Methods and compositions for preventing immune suppression are also disclosed. The methods involve administering an effective amount of an agent that inhibits OX-2.

[0001] This application is a continuation-in-part of U.S. applicationSer. No. 09/570,367 filed May 5, 1998 (now allowed) which is acontinuation of PCT/CA98/01038 filed Nov. 6, 1998 (which designated theU.S.) which claims the benefit of U.S. Ser. No. 60/064,764 filed Nov. 7,1997 (now abandoned).

FIELD OF THE INVENTION

[0002] The present invention relates to methods and compositions formodulating an immune response. The invention includes the use of theprotein OX-2 to suppress an immune response and to prevent fetal loss.

BACKGROUND OF THE INVENTION

[0003] The immune system protects the body from infectious agents anddisease and is critical to our survival. However, in certain instances,the immune system can be the cause of illness. One example is inautoimmune disease wherein the immune system attacks its own hosttissues, in many instances causing debilitating illness and sometimesresulting in death. Examples of autoimmune diseases include multiplesclerosis, type 1 insulin-dependent diabetes mellitus, lupuserythematosus and arthritis. A second example where the immune systemcan cause illness is during tissue or organ transplantation. Except inthe cases of genetically identical animals, such as monozygotic twins,tissue and organ transplants are rejected by the recipient's immunesystem as foreign. The immune reaction against transplants is even morepronounced in transplantation across species or xenotransplantation. Athird example where the immune system harms the host is during anallergic reaction where the immune system is activated by a generallyinnocuous antigen causing inflammation and in some cases tissue damage.

[0004] In order to inhibit the detrimental immune reactions duringtransplantation, autoimmune disease and allergic reactions,immunosuppressive drugs (such as cyclosporin A, tacrolimas, andcorticosteroids) or antibody therapies (such as anti-T cell antibodies)are generally administered. Unfortunately, these non-specific modes ofimmunosuppression generally have undesirable side effects. For example,cyclosporin may cause decreased renal function, hypertension, toxicityand it must be administered for the life of the patient. Corticosteroidsmay cause decreased resistance to infection, painful arthritis,osteoporosis and cataracts. The anti-T cell antibodies may cause fever,hypertension, diarrhea or sterile meningitis and are quite expensive.

[0005] In view of the problems associated with immunosuppression, therehas been an interest in developing methods or therapies that induceunresponsiveness or tolerance in the host to a transplant, to “self”tissues in autoimmune disease and to harmless antigens associated withallergies. The inventor has been studying the mechanisms involved intransplant rejection and has developed methods for inducing a state ofantigen-specific immunological tolerance in transplantation. Inparticular, in animal allograft models, the inventor has demonstratedthat graft survival can been increased if the recipient animal is givena pre-transplant infusion via the portal vein of irradiated spleen cellsfrom the donor animal. In contrast, a pre-transplant infusion via thetail vein does not prolong graft survival.

[0006] Understanding the molecular mechanisms involved in the inductionof tolerance following portal-venous (pv) immunization may lead to thedevelopment of methods of inducing immune tolerance that may be usefulin transplant, autoimmune disease and allergies.

SUMMARY OF THE INVENTION

[0007] Genes that show an increase in expression following portal venousimmunization have been identified. One of the genes isolated encodesOX-2, a molecule with previously unknown function belonging to the Igsuperfamily. The OX-2 molecule is also generally referred to as CD200 inthe current literature. The inventors have shown that administeringantibodies to OX-2 inhibited the graft survival generally seen followingpre-transplant pv immunization. The inventors have also shown that thereis a negative association between levels of OX-2 and risk of fetal loss.In particular, the inventors have shown administering OX-2 reduced fetalloss rates while inhibiting OX-2 reversed the effect. The inventors havefurther shown that OX-2 inhibits cytotoxic cells and IL-2 production andinduces IL-4 production. All of these results demonstrate that OX-2 isinvolved in immune suppression.

[0008] Consequently, broadly stated, the present invention provides amethod of suppressing an immune system comprising administering aneffective amount of an OX-2 protein or a nucleic acid sequence encodingan OX-2 protein to an animal in need of such treatment.

[0009] In one embodiment, the present invention provides a method ofpreventing or inhibiting fetal loss comprising administering aneffective amount of an OX-2 protein or a nucleic acid sequence encodingan OX-2 protein to an animal in need thereof.

[0010] The invention also includes pharmaceutical compositionscontaining OX-2 proteins or nucleic acids encoding OX-2 proteins for usein inducing tolerance in transplantation or autoimmune disease.

[0011] As stated above, OX-2 can be used to induce immune suppression.Consequently, inhibiting OX-2 may also be useful in preventing immunesuppression.

[0012] Therefore, in another aspect, the present invention provides amethod of preventing immune suppression comprising administering aneffective amount of an agent that inhibits OX-2 to an animal in needthereof. In a preferred embodiment the OX-2 inhibitor is an antibodythat binds OX-2 or an antisense oligonucleotide that inhibits theexpression of OX-2.

[0013] In one embodiment, the present invention provides a method ofinducing fetal loss comprising administering an effective amount of anagent that inhibits OX-2 to an animal in need thereof.

[0014] The invention also includes pharmaceutical compositionscontaining an OX-2 inhibitor for use in inducing or augmenting an immuneresponse.

[0015] Other features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating preferred embodiments of the invention aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The invention will now be described in relation to the drawingsin which:

[0017]FIG. 1 illustrates PCR validation of suppressive subtractivehybridization using β-actin primers.

[0018]FIG. 2 illustrates PCR validation of suppressive subtractivehybridization using IL-10 primers.

[0019]FIG. 3 is an autoradiograph using ³²P-labeled probes from 4 clonesobtained from the subtractive hybridization process.

[0020]FIG. 4 is flow cytometry profile of spleen adherent cells.

[0021]FIG. 5A and B are Western Blots illustrating the increasedexpression of OX-2 antigen after pv immunization. FIG. 5A shows stainingwith a control mouse antibody, anti-mouse CD8a. FIG. 5B shows stainingwith anti-rat MRC OX-2.

[0022]FIG. 6 is a graft showing percent survival versus days post renaltransplantation.

[0023]FIG. 7 shows the cDNA sequence of rat (SEQ.ID.NO.:20), mouse(SEQ.ID.NO.:22) and human MRC OX-2 (SEQ.ID.NO.:18).

[0024]FIG. 8 shows the deduced protein sequence of rat (SEQ.ID.NO.:21),mouse (SEQ.ID.NO.:2) and human MRC OX-2 (SEQ.ID.NO.:19) protein.

[0025]FIGS. 9A and 9B are bar graphs showing cytokine production andcell proliferation following stimulation by allogeneic DC using hepaticNPMC.

[0026]FIGS. 10A, 10B and 10C are bar graphs showing inhibition of cellproliferation and cytokine production by hepatic NPMC.

[0027]FIG. 11 is a bar graph analysis of FACS data showing OX-2expression in a subpopulation of NPC.

[0028]FIG. 12 shows PCR analysis mRNA expression of B7-1, B7-2 and OX-2in various hepatic NPMC cell fractions.

[0029]FIGS. 13A and 13B are bar graphs showing proliferation andcytokine production by NPMC from F/t3L treated mice.

[0030]FIG. 14 is a bar graph showing cytokines produced from C3H micewith C57BL16 renal allografts and NPC from Flt3 treated C57BL16 donors.

[0031]FIG. 15 is a graph showing inhibition of graft rejection with NPCfrom Flt3 treated mice.

[0032]FIG. 16 is a graph showing that anti-OX-2 reverses inhibition byNPC. The effect of anti-B7-1, anti-B7-2 and anti-OX-2 on primaryallostimulation is shown.

[0033]FIG. 17 is a graph showing that anti-OX-2 mAb reverses inhibitionby NPC and inhibits the development of immunoregulatory cells.

[0034]FIG. 18A is a photograph showing in situ hybridization withantisense OX-2 in a day 8.5 implantation site from a mouse CBA/JxDBA/2that is susceptible to fetal loss.

[0035]FIG. 18B is a photograph showing in situ hybridization withantisense OX-2 in a day 8.5 implantation site from a mouse DBA/2XCBA/Jthat is not susceptible to spontaneous fetal loss.

[0036]FIG. 19 is a graph showing the effect of anti-OX-2 monoclonalantibody 3B6 on spontaneous resorption (abortion) rates. Error bars show1 standard deviation. A minimum of 20 implants was scored for eachgroup. * significant increase in abortion rate, P<0.02.

[0037]FIG. 20 is a graph showing effect of OX2 immunoadhesion (OX2:Fc)on spontaneous resorption (abortion) rates or renal allograft rejection.Error bars show 1 standard deviation. A minimum of 45 implants wasscored for each group of pregnant mice (mice received a single dose ofOX2:Fc at day 6.5; 10 mice/group were used for renal allografts, andmice received multiple iv injections of OX2:Fc, beginning on the day oftransplant, and at 2 day intervals thereafter. * significant decrease inabortion rate, or increased graft survival, relative to controls.

[0038]FIG. 21 shows the importance of CD200 (OX-2) in rescuingpotentially doomed embryos which are low in OX-2 mRNA expression by thein situ hybridisation result.

[0039]FIG. 22 shows the molecule size ladder (lane 1), trophoblast withfull length primers (lane 2) and exon 3 primers (lane 3). The fulllength and shorter mRNA OX-2 transcripts are seen. Lane 4 (correspondingto lane 2) and lane 5 (corresponding to lane 3) represent negativeresult obtained with CD9⁺ stromal cells.

[0040]FIG. 23 shows result using antibody detecting CK5,6,8,17; similarresults have been obtained using FITC-anti-CK18. The upper left panelshows the forward and side scatter pattern. FITC and PE isotype controlsused to set gates is shown below on left. Upper right panel showscytokeratin-positive cells, the majority of which were also OX-2⁺. Asignificant population of CK− OX-2⁺⁺ cells was also noted. The identityof this population has not been established. On the right, the middlepanel shows staining profile for OX-2 for the CK− and CK⁺ populations,and the lower panel shows the scatter. Clearly these populations havedifferent properties. Velocity sedimentation separation indicates bothpopulations may have OX-2-dependent immunoregulatory activity.

DETAILED DESCRIPTION OF THE INVENTION

[0041] The present inventors have identified genes that show an increasein expression following portal venous immunization. These genes play arole in the development of immune suppression or tolerance and may beuseful in developing therapies for the prevention and treatment oftransplant rejection, fetal loss, autoimmune disease or allergies.

[0042] Using suppression subtractive hybridization (SSH), the inventorhas isolated a clone that is preferentially expressed in mice receivingallogenic renal grafts along with pre-transplant donor-specificimmunization and that encodes the protein OX-2. The OX-2 protein (alsoknown as MRC OX-2) in rat was described as a 41 Kd-47 Kd glycoproteinwhich is expressed on the cell surface of thymocytes, folliculardendritic cells and endothelium, B cells and neuronal cells. Differencesin apparent size of the molecule in different tissues is probably afunction of differential glycosylation. The function of the molecule waspreviously unknown, but DNA and amino acid sequence analysis shows ithas a high degree of homology to molecules of the immunoglobulin genefamily, which includes molecules important in lymphocyte antigenrecognition and cell-cell interaction (e.g. CD4, CD8, ICAMs, VCAMs), aswell as adhesion receptor molecules (NCAMs) in the nervous system.Members of the immunoglobulin superfamily are distinct from othermolecules of the integrin and selectin families, which, at least withinthe immune system, also seem to play critical role in cell recognition,migration and even development of the lymphocyte recognition repertoire(by regulating intra-thymic selection events). It has becomeincreasingly evident that molecules of these different families play animportant role in human disease.

[0043] The inventor has shown that administering antibodies to OX-2inhibited the graft survival generally seen following pre-transplant pvimmunization. The inventor has also shown that there is negativeassociation between levels of OX-2 and risk of fetal loss and thatadministering OX-2 prevents fetal loss and inhibiting OX-2 causes fetalloss. The inventor has further shown that OX-2 inhibits cytotoxic cellsand IL-2 production and induces IL-4 production. The data supports therole of OX-2 in immune suppression.

Therapeutic Methods Inducing Immune Suppression

[0044] In one aspect, the present invention provides a method ofsuppressing an immune response comprising administering an effectiveamount of an OX-2 protein or a nucleic acid sequence encoding an OX-2protein to an animal in need of such treatment. The invention includes ause of an effective amount of an OX-2 protein or a nucleic acid sequenceencoding an OX-2 protein to suppress an immune response.

[0045] The term “OX-2 protein” includes OX-2 or CD200 from any speciesor source and includes a full length OX-2 protein as well as fragmentsor portions of the protein. The term “OX-2” is also generally referredto as “CD200” due to a change in nomenclature. Both “OX-2” and “CD200”may be used interchangeably in the application. Preferred fragments orportions of the OX-2 or CD200 protein are those that are sufficient tosuppress an immune response. Determining whether a particular OX-2 orCD200 protein can suppress an immune response can be assessing usingknown in vitro immune assays including, but not limited to, inhibiting amixed leucocyte reaction; inhibiting a cytotoxic T cell response;inhibiting interleukin-2 production; inhibiting IFNγ production;inhibiting a Th1 cytokine profile; inducing IL-4 production; inducingTGFβ production; inducing IL-10 production; inducing a Th2 cytokineprofile; and any other assay that would be known to one of skill in theart to be useful in detecting immune suppression.

[0046] One of skill in the art can also determine whether or not aparticular OX-2 or fragment thereof is useful in preventing fetal loss.As mentioned above, one of skill in the art can readily test an OX-2 orOX-2 fragment for its ability to suppress an immune response using knownin vitro assays. In addition the OX-2 or OX-2 fragment can also betested for its ability to prevent fetal loss in an animal model. Forexample, one could use the model described in Example 8 wherein theability of OX-2 to prevent cytokine induced abortion in abortion-proneCBA x DBA/2 mice is assessed. Further, mice pre-immunized withanti-phospholipid may also be used.

[0047] The term “administering an OX-2 protein” includes both theadministration of the OX-2 protein as well as the administration of anucleic acid sequence encoding an OX-2 protein. In the latter case, theOX-2 protein is produced in vivo in the animal.

[0048] In a preferred embodiment, the OX-2 protein is prepared andadministered as a soluble fusion protein. The fusion protein may containthe extracellular domain of OX-2 linked to an immunoglobulin (Ig) FcRegion. The OX-2 fusion may be prepared using techniques known in theart. Generally, a DNA sequence encoding the extracellular domain of OX-2is linked to a DNA sequence encoding the Fc of the lg and expressed inan appropriate expression system where the OX-2-Fclg fusion protein isproduced.

[0049] The OX-2 or protein may be obtained from known sources orprepared using recombinant DNA techniques. The protein may have any ofthe known published sequences for OX-2 or CD200. The sequences can beobtained from GenBank. The human sequence has accession no. M17226X0523; the rat sequence has accession no. X01785; and the mouse sequencehas accession no. AF029214. The nucleic acid and protein sequences ofOX-2 (CD200) from human, mouse and rat are also shown in FIGS. 7 and 8and in SEQ.ID.Nos.: 18, 22 and 20 (nucleic acid) and SEQ.ID.Nos.:19, 21and 2 (protein).

[0050] The OX-2 protein may also be modified to contain amino acidsubstitutions, insertions and/or deletions that do not alter theimmunosuppressive properties of the protein. Conserved amino acidsubstitutions involve replacing one or more amino acids of the OX-2amino acid sequence with amino acids of similar charge, size, and/orhydrophobicity characteristics. When only conserved substitutions aremade the resulting analog should be functionally equivalent to the OX-2protein. Non-conserved substitutions involve replacing one or more aminoacids of the OX-2 amino acid sequence with one or more amino acids whichpossess dissimilar charge, size, and/or hydrophobicity characteristics.

[0051] The OX-2 protein may be modified to make it more therapeuticallyeffective or suitable. For example, the OX-2 protein may be cyclized ascyclization allows a peptide to assume a more favourable conformation.Cyclization of the OX-2 peptides may be achieved using techniques knownin the art. In particular, disulphide bonds may be formed between twoappropriately spaced components having free sulfhydryl groups. The bondsmay be formed between side chains of amino acids, non-amino acidcomponents or a combination of the two. In addition, the OX-2 protein orpeptides of the present invention may be converted into pharmaceuticalsalts by reacting with inorganic acids including hydrochloric acid,sulphuric acid, hydrobromic acid, phosphoric acid, etc., or organicacids including formic acid, acetic acid, propionic acid, glycolic acid,lactic acid, pyruvic acid, oxalic acid, succinic acid, malic acid,tartaric acid, citric acid, benzoic acid, salicylic acid,benzenesulphonic acid, and tolunesulphonic acids.

[0052] Administration of an “effective amount” of the OX-2 protein andnucleic acid of the present invention is defined as an amount effective,at dosages and for periods of time necessary to achieve the desiredresult. The effective amount of the OX-2 protein or nucleic acid of theinvention may vary according to factors such as the disease state, age,sex, and weight of the animal. Dosage regima may be adjusted to providethe optimum therapeutic response. For example, several divided doses maybe administered daily or the dose may be proportionally reduced asindicated by the exigencies of the therapeutic situation.

[0053] The term “animal” as used herein includes all members of theanimal kingdom including humans. When treating fetal loss, the animal isa female who is desirous of becoming pregnant or maintaining apregnancy.

[0054] The present inventors have shown that there is an associationbetween levels of OX-2 expression and fertility. In particular theinventor has shown that low levels (or no levels) of OX-2 is related tofetal loss. Further, administering a OX-2:Fc fusion protein preventedfetal loss.

[0055] Accordingly, the present invention provides a method ofpreventing, reducing or inhibiting fetal loss comprising administeringan effective amount of an OX-2 protein or a nucleic acid sequenceencoding an OX-2 protein to an animal in need thereof. The inventionincludes a use of an effective amount of an OX-2 protein on a nucleicacid molecules encoding an OX-2 protein to prevent or inhibit fetalloss. The OX-2 protein may be from any species and may be the fulllength sequence or a fragment thereof that is capable of preventing orinhibiting fetal loss.

[0056] In another embodiment, the present invention provides a method ofinducing immune tolerance to a transplanted organ or tissue in arecipient animal comprising administering an effective amount of a OX-2protein or a nucleic acid sequence encoding an OX-2 protein to therecipient animal prior to the transplantation of the organ or tissue.The invention includes a use of an effective amount of a OX-2 protein ora nucleic acid sequence encoding an OX-2 protein to induce immunetolerance to a transplanted organ or tissue.

[0057] The term “inducing immune tolerance” means rendering the immunesystem unresponsive to a particular antigen without inducing a prolongedgeneralized immune deficiency. The term “antigen” means a substance thatis capable of inducing an immune response. In the case of autoimmunedisease, immune tolerance means rendering the immune system unresponsiveto an auto-antigen that the host is recognizing as foreign, thus causingan autoimmune response. In the case of allergy, immune tolerance meansrendering the immune system unresponsive to an allergen that generallycauses an immune response in the host. In the case of transplantation,immune tolerance means rendering the immune system unresponsive to theantigens on the transplant. An alloantigen refers to an antigen foundonly in some members of a species, such as blood group antigens. Axenoantigen refers to an antigen that is present in members of onespecies but not members of another. Correspondingly, an allograft is agraft between members of the same species and a xenograft is a graftbetween members of a different species.

[0058] The recipient can be any member of the animal kingdom includingrodents, pigs, cats, dogs, ruminants, non-human primates and preferablyhumans. The organ or tissue to be transplanted can be from the samespecies as the recipient (allograft) or can be from another species(xenograft). The tissues or organs can be any tissue or organ includingheart, liver, kidney, lung, pancreas, pancreatic islets, brain tissue,cornea, bone, intestine, skin and heamatopoietic cells.

[0059] The method of the invention may be used to prevent graft versushost disease wherein the immune cells in the transplant mount an immuneattack on the recipient's immune system. This can occur when the tissueto be transplanted contains immune cells such as when bone marrow orlymphoid tissue is transplanted when treating leukemias, aplasticanemias and enzyme or immune deficiencies, for example.

[0060] Accordingly, in another embodiment, the present inventionprovides a method of preventing or inhibiting graft versus host diseasein a recipient animal receiving an organ or tissue transplant comprisingadministering an effective amount of a OX-2 protein or a nucleic acidsequence encoding an OX-2 protein to the organ or tissue prior to thetransplantation in the recipient animal. The invention includes a use ofan effective amount of an OX-2 protein or a nucleic acid moleculeencoding an OX-2 protein to prevent or inhibit graft versus hostdisease.

[0061] As stated previously, the method of the present invention mayalso be used to treat or prevent autoimmune disease. In an autoimmunedisease, the immune system of the host fails to recognize a particularantigen as “self” and an immune reaction is mounted against the host'stissues expressing the antigen. Normally, the immune system is tolerantto its own host's tissues and autoimmunity can be thought of as abreakdown in the immune tolerance system.

[0062] Accordingly, in a further embodiment, the present inventionprovides a method of preventing or treating an autoimmune diseasecomprising administering an effective amount of an OX-2 protein or anucleic acid sequence encoding an OX-2 protein to an animal having,suspected of having, or susceptible to having an autoimmune disease. Theinvention includes a use of an effective amount of an OX-2 protein on anucleic acid molecule encoding an OX-2 protein to prevent or inhibit anautoimmune disease.

[0063] Autoimmune diseases that may be treated or prevented according tothe present invention include, but are not limited to, type 1insulin-dependent diabetes mellitus, adult respiratory distresssyndrome, inflammatory bowel disease, dermatitis, meningitis, thromboticthrombocytopenic purpura, Sjogren's syndrome, encephalitis, uveitic,leukocyte adhesion deficiency, rheumatoid arthritis, rheumatic fever,Reiter's syndrome, psoriatic arthritis, progressive systemic sclerosis,primary biniary cirrhosis, pemphigus, pemphigoid, necrotizingvasculitis, myasthenia gravis, multiple sclerosis, lupus erythematosus,polymyositis, sarcoidosis, granulomatosis, vasculitis, perniciousanemia, CNS inflammatory disorder, antigen-antibody complex mediateddiseases, autoimmune haemolytic anemia, Hashimoto's thyroiditis, Gravesdisease, habitual spontaneous abortions, Reynard's syndrome,glomerulonephritis, dermatomyositis, chronic active hepatitis, celiacdisease, autoimmune complications of AIDS, atrophic gastritis,ankylosing spondylitis and Addison's disease.

[0064] As stated previously, the method of the present invention mayalso be used to treat or prevent an allergic reaction. In an allergicreaction, the immune system mounts an attack against a generallyharmless, innocuous antigen or allergen. Allergies that may be preventedor treated using the methods of the invention include, but are notlimited to, hay fever, asthma, atopic eczema as well as allergies topoison oak and ivy, house dust mites, bee pollen, nuts, shellfish,penicillin and numerous others.

[0065] Accordingly, in a further embodiment, the present inventionprovides a method of preventing or treating an allergy comprisingadministering an effective amount of an OX-2 protein or a nucleic acidsequence encoding an OX-2 protein to an animal having or suspected ofhaving an allergy. The invention includes a use of an effective amountof an OX-2 protein or a nucleic acid molecule encoding an OX-2 proteinto prevent or treat an allergy.

Preventing Immune Suppression

[0066] In another aspect, the present invention provides a method ofpreventing immune suppression comprising administering an effectiveamount of an agent that inhibits OX-2 to an animal in need thereof.

[0067] There are a large number of situations whereby it is desirable toprevent immune suppression including, but not limited to, the treatmentof infections, cancer and Acquired Immune Deficiency Syndrome and theinduction of fetal loss.

[0068] Accordingly, the present invention provides a method ofpreventing immune suppression comprising administering an effectiveamount of an agent that inhibits OX-2 to an animal in need thereof. Inone embodiment the present invention provides a method of inducing fetalloss comprising administering an effective amount of an agent thatinhibits OX-2 to an animal in need thereof. The animal is preferably apregnant female.

[0069] The agent that inhibits OX-2 can be any agent that decreases theexpression or activity of an OX-2 protein such that the immunesuppression caused by OX-2 is reduced, inhibited and/or prevented. Oneof skill in the art can readily determine whether or not a particularagent is effective in inhibiting OX-2. For example, the agent can betested in in vitro assays to determine if the function or activity ofOX-2 is inhibited. The agent can also be tested for its ability toinduce an immune response using in vitro immune assays including, butnot limited to, enhancing a cytotoxic T cell response; inducinginterleukin-2 (IL-2) production; inducing IFNγ production; inducing aTh1 cytokine profile; inhibiting IL-4 production; inhibiting TGFβproduction; inhibiting IL-10 production; inhibiting a Th2 cytokineprofile and any other assay that would be known to one of skill in theart to be useful in detecting immune activation.

[0070] One of skill in the art can also determine whether a particularagent is useful in inducing fetal loss. As mentioned above, one can testthe agent for its ability to induce an immune response using known invitro assays. In addition, the agent can be tested in an animal model,for example as described in Example 11, wherein the agent isadministered to a pregnant rodent.

[0071] In a preferred embodiment, the agent that inhibits OX-2 is anOX-2 specific antibody. The present inventor has prepared antibodies toOX-2 which are described in Examples 4 and 5. Antibodies to OX-2 mayalso be prepared using techniques known in the art such as thosedescribed by Kohler and Milstein, Nature 256, 495 (1975) and in U.S.Pat. Nos. RE 32,011; 4,902,614; 4,543,439; and 4,411,993, which areincorporated herein by reference. (See also Monoclonal Antibodies,Hybridomas: A New Dimension in Biological Analyses, Plenum Press,Kennett, McKearn, and Bechtol (eds.), 1980, and Antibodies: A LaboratoryManual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press,1988, which are also incorporated herein by reference). Within thecontext of the present invention, antibodies are understood to includemonoclonal antibodies, polyclonal antibodies, antibody fragments (e.g.,Fab, and F(ab′)₂) and recombinantly produced binding partners.

[0072] Accordingly, the present invention provides a method ofpreventing immune suppression or inducing fetal loss comprisingadministering an effective amount of an antibody that inhibits OX-2 toan animal in need thereof.

[0073] In another embodiment, the OX-2 inhibitor is an antisenseoligonucleotide that inhibits the expression of OX-2. Antisenseoligonucleotides that are complimentary to a nucleic acid sequence froman OX-2 gene can be used in the methods of the present invention toinhibit OX-2. The present inventor has prepared antisenseoligonucleotides to OX-2 which are described in Example 3.

[0074] Accordingly, the present invention provides a method ofpreventing immune suppression comprising administering an effectiveamount of an antisense oligonucleotide that is complimentary to anucleic acid sequence from a OX-2 gene to an animal in need thereof.

[0075] The term antisense oligonucleotide as used herein means anucleotide sequence that is complimentary to its target.

[0076] In one embodiment of the invention, the present inventionprovides an antisense oligonucleotide that is complimentary to a nucleicacid molecule having a sequence as shown in FIG. 7, wherein T can alsobe U, or a fragment thereof.

[0077] The term “oligonucleotide” refers to an oligomer or polymer ofnucleotide or nucleoside monomers consisting of naturally occurringbases, sugars, and intersugar (backbone) linkages. The term alsoincludes modified or substituted oligomers comprising non-naturallyoccurring monomers or portions thereof, which function similarly. Suchmodified or substituted oligonucleotides may be preferred over naturallyoccurring forms because of properties such as enhanced cellular uptake,or increased stability in the presence of nucleases. The term alsoincludes chimeric oligonucleotides which contain two or more chemicallydistinct regions. For example, chimeric oligonucleotides may contain atleast one region of modified nucleotides that confer beneficialproperties (e.g. increased nuclease resistance, increased uptake intocells), or two or more oligonucleotides of the invention may be joinedto form a chimeric oligonucleotide.

[0078] The antisense oligonucleotides of the present invention may beribonucleic or deoxyribonucleic acids and may contain naturallyoccurring bases including adenine, guanine, cytosine, thymidine anduracil. The oligonucleotides may also contain modified bases such asxanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl and otheralkyl adenines, 5-halo uracil, 5-halo cytosine, 6-aza uracil, 6-azacytosine and 6-aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine,8-aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines, 8-hydroxyladenine and other 8-substituted adenines, 8-halo guanines, 8-aminoguanine, 8-thiol guanine, 8-thiolalkyl guanines, 8-hydroxyl guanine andother 8-substituted guanines, other aza and deaza uracils, thymidines,cytosines, adenines, or guanines, 5-trifluoromethyl uracil and5-trifluoro cytosine.

[0079] Other antisense oligonucleotides of the invention may containmodified phosphorous, oxygen heteroatoms in the phosphate backbone,short chain alkyl or cycloalkyl intersugar linkages or short chainheteroatomic or heterocyclic intersugar linkages. For example, theantisense oligonucleotides may contain phosphorothioates,phosphotriesters, methyl phosphonates, and phosphorodithioates. In anembodiment of the invention there are phosphorothioate bonds linksbetween the four to six 3′-terminus bases. In another embodimentphosphorothioate bonds link all the nucleotides.

[0080] The antisense oligonucleotides of the invention may also comprisenucleotide analogs that may be better suited as therapeutic orexperimental reagents. An example of an oligonucleotide analogue is apeptide nucleic acid (PNA) wherein the deoxyribose (or ribose) phosphatebackbone in the DNA (or RNA), is replaced with a polyamide backbonewhich is similar to that found in peptides (P. E. Nielsen, et al Science1991, 254, 1497). PNA analogues have been shown to be resistant todegradation by enzymes and to have extended lives in vivo and in vitro.PNAs also bind stronger to a complimentary DNA sequence due to the lackof charge repulsion between the PNA strand and the DNA strand. Otheroligonucleotides may contain nucleotides containing polymer backbones,cyclic backbones, or acyclic backbones. For example, the nucleotides mayhave morpholino backbone structures (U.S. Pat. No. 5,034,506).Oligonucleotides may also contain groups such as reporter groups, agroup for improving the pharmacokinetic properties of anoligonucleotide, or a group for improving the pharmacodynamic propertiesof an antisense oligonucleotide. Antisense oligonucleotides may alsohave sugar mimetics.

[0081] The antisense nucleic acid molecules may be constructed usingchemical synthesis and enzymatic ligation reactions using proceduresknown in the art. The antisense nucleic acid molecules of the inventionor a fragment thereof, may be chemically synthesized using naturallyoccurring nucleotides or variously modified nucleotides designed toincrease the biological stability of the molecules or to increase thephysical stability of the duplex formed with mRNA or the native genee.g. phosphorothioate derivatives and acridine substituted nucleotides.The antisense sequences may be produced biologically using an expressionvector introduced into cells in the form of a recombinant plasmid,phagemid or attenuated virus in which antisense sequences are producedunder the control of a high efficiency regulatory region, the activityof which may be determined by the cell type into which the vector isintroduced.

Compositions

[0082] The invention also includes pharmaceutical compositionscontaining OX-2 proteins or nucleic acids for use in immune suppressionas well as pharmaceutical compositions containing an OX-2 inhibitor foruse in preventing immune suppression.

[0083] Such pharmaceutical compositions can be for intralesional,intravenous, topical, rectal, parenteral, local, inhalant orsubcutaneous, intradermal, intramuscular, intrathecal, transperitoneal,oral, and intracerebral use. The composition can be in liquid, solid orsemisolid form, for example pills, tablets, creams, gelatin capsules,capsules, suppositories, soft gelatin capsules, gels, membranes,tubelets, solutions or suspensions.

[0084] The pharmaceutical compositions of the invention can be intendedfor administration to humans or animals. Dosages to be administereddepend on individual needs, on the desired effect and on the chosenroute of administration.

[0085] The pharmaceutical compositions can be prepared by per se knownmethods for the preparation of pharmaceutically acceptable compositionswhich can be administered to patients, and such that an effectivequantity of the active substance is combined in a mixture with apharmaceutically acceptable vehicle. Suitable vehicles are described,for example, in Remington's Pharmaceutical Sciences (Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA1985).

[0086] On this basis, the pharmaceutical compositions include, albeitnot exclusively, the active compound or substance in association withone or more pharmaceutically acceptable vehicles or diluents, andcontained in buffered solutions with a suitable pH and iso-osmotic withthe physiological fluids. The pharmaceutical compositions mayadditionally contain other agents such as immunosuppressive drugs orantibodies to enhance immune tolerance or immunostimulatory agents toenhance the immune response.

[0087] In one embodiment, the pharmaceutical composition for use inpreventing fetal loss comprises an effective amount of a OX-2 protein inadmixture with a pharmaceutically acceptable diluent or carrier. TheOX-2 protein is preferably prepared as an immunoadhesion molecule insoluble form which can be administered to the patient.

[0088] In another embodiment, the pharmaceutical composition for use inpreventing fetal loss comprises an effective amount of a nucleic acidmolecule encoding a OX-2 protein in admixture with a pharmaceuticallyacceptable diluent or carrier.

[0089] The nucleic acid molecules of the invention encoding a OX-2protein may be used in gene therapy to induce immune tolerance.Recombinant molecules comprising a nucleic acid sequence encoding a OX-2protein, or fragment thereof, may be directly introduced into cells ortissues in vivo using delivery vehicles such as retroviral vectors,adenoviral vectors and DNA virus vectors. They may also be introducedinto cells in vivo using physical techniques such as microinjection andelectroporation or chemical methods such as coprecipitation andincorporation of DNA into liposomes. Recombinant molecules may also bedelivered in the form of an aerosol or by lavage. The nucleic acidmolecules of the invention may also be applied extracellularly such asby direct injection into cells. The nucleic acid molecules encoding OX-2are preferably prepared as a fusion with a nucleic acid moleculeencoding an immunoglobulin (Ig) Fc region. As such, the OX-2 proteinwill be expressed in vivo as a soluble fusion protein.

[0090] In another aspect, the pharmaceutical composition for use ininducing fetal loss comprises an effective amount of a OX-2 inhibitor inadmixture with a pharmaceutically acceptable diluent or carrier. Suchcompositions may be administered as a vaccine either alone or incombination with other active agents.

[0091] In one embodiment, the pharmaceutical composition for use ininducing fetal loss comprises an effective amount of an antibody to OX-2in admixture with a pharmaceutically acceptable diluent or carrier. Theantibodies may be delivered intravenously.

[0092] In another embodiment, the pharmaceutical composition for use ininducing fetal loss comprises an effective amount of an antisenseoligonucleotide nucleic acid complimentary to a nucleic acid sequencefrom a OX-2 gene in admixture with a pharmaceutically acceptable diluentor carrier. The oligonucleotide molecules may be administered asdescribed above for the compositions containing OX-2 nucleic acidsequences.

Murine OX-2

[0093] The inventor has cloned and sequenced the murine OX-2 gene.Accordingly, the invention also includes an isolated nucleic acidsequence encoding a murine OX-2 gene and having the sequence shown inFIG. 7 and SEQ.ID.NO.:1.

[0094] The term “isolated” refers to a nucleic acid substantially freeof cellular material or culture medium when produced by recombinant DNAtechniques, or chemical precursors, or other chemicals when chemicallysynthesized. The term “nucleic acid” is intended to include DNA and RNAand can be either double stranded or single stranded.

[0095] Preferably, the purified and isolated nucleic acid molecule ofthe invention comprises (a) a nucleic acid sequence as shown inSEQ.ID.NO.:1, wherein T can also be U; (b) nucleic acid sequencescomplementary to (a); (c) a fragment of (a) or (b) that is at least 15bases, preferably 20 to 30 bases, and which will hybridize to (a) or (b)under stringent hybridization conditions; or (a) a nucleic acid moleculediffering from any of the nucleic acids of (a) or (b) in codon sequencesdue to the degeneracy of the genetic code.

[0096] It will be appreciated that the invention includes nucleic acidmolecules encoding truncations of the murine OX-2 proteins of theinvention, and analogs and homologs of the proteins of the invention andtruncations thereof, as described below. It will further be appreciatedthat variant forms of the nucleic acid molecules of the invention whicharise by alternative splicing of an mRNA corresponding to a cDNA of theinvention are encompassed by the invention.

[0097] An isolated nucleic acid molecule of the invention which is DNAcan also be isolated by selectively amplifying a nucleic acid encoding anovel protein of the invention using the polymerase chain reaction (PCR)methods and cDNA or genomic DNA. It is possible to design syntheticoligonucleotide primers from the nucleic acid molecules as shown in FIG.7 for use in PCR. A nucleic acid can be amplified from cDNA or genomicDNA using these oligonucleotide primers and standard PCR amplificationtechniques. The nucleic acid so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis. It willbe appreciated that cDNA may be prepared from mRNA, by isolating totalcellular mRNA by a variety of techniques, for example, by using theguanidinium-thiocyanate extraction procedure of Chirgwin et al.,Biochemistry, 18, 5294-5299 (1979). cDNA is then synthesized from themRNA using reverse transcriptase (for example, Moloney MLV reversetranscriptase available from Gibco/BRL, Bethesda, Md., or AMV reversetranscriptase available from Seikagaku America, Inc., St. Petersburg,Fla.).

[0098] An isolated nucleic acid molecule of the invention which is RNAcan be isolated by cloning a cDNA encoding a novel protein of theinvention into an appropriate vector which allows for transcription ofthe cDNA to produce an RNA molecule which encodes a OX-2 protein of theinvention. For example, a cDNA can be cloned downstream of abacteriophage promoter, (e.g. a T7 promoter) in a vector, cDNA can betranscribed in vitro with T7 polymerase, and the resultant RNA can beisolated by standard techniques.

[0099] A nucleic acid molecule of the invention may also be chemicallysynthesized using standard techniques. Various methods of chemicallysynthesizing polydeoxynucleotides are known, including solid-phasesynthesis which, like peptide synthesis, has been fully automated incommercially available DNA synthesizers (See e.g., Itakura et al. U.S.Pat. No. 4,598,049; Caruthers et al. U.S. Pat. No. 4,458,066; andItakura U.S. Pat. Nos. 4,401,796 and 4,373,071).

[0100] The sequence of a nucleic acid molecule of the invention may beinverted relative to its normal presentation for transcription toproduce an antisense nucleic acid molecule. Preferably, an antisensesequence is constructed by inverting a region preceding the initiationcodon or an unconserved region. In particular, the nucleic acidsequences contained in the nucleic acid molecules of the invention or afragment thereof, preferably a nucleic acid sequence shown in FIG. 7 maybe inverted relative to its normal presentation for transcription toproduce antisense nucleic acid molecules.

[0101] The antisense nucleic acid molecules of the invention or afragment thereof, may be chemically synthesized using naturallyoccurring nucleotides or variously modified nucleotides designed toincrease the biological stability of the molecules or to increase thephysical stability of the duplex formed with mRNA or the native genee.g. phosphorothioate derivatives and acridine substituted nucleotides.The antisense sequences may be produced biologically using an expressionvector introduced into cells in the form of a recombinant plasmid,phagemid or attenuated virus in which antisense sequences are producedunder the control of a high efficiency regulatory region, the activityof which may be determined by the cell type into which the vector isintroduced.

[0102] The invention also provides nucleic acids encoding fusionproteins comprising a OX-2 protein of the invention and a selectedprotein, or a selectable marker protein.

[0103] The invention further includes an isolated protein which has theamino acid sequence as shown in FIG. 8 and SEQ.ID.NO.:2.

[0104] Within the context of the present invention, a protein of theinvention may include various structural forms of the primary proteinwhich retain biological activity. For example, a protein of theinvention may be in the form of acidic or basic salts or in neutralform. In addition, individual amino acid residues may be modified byoxidation or reduction.

[0105] In addition to the full length amino acid sequence (FIG. 8), theprotein of the present invention may also include truncations of theprotein, and analogs, and homologs of the protein and truncationsthereof as described herein. Truncated proteins may comprise peptides ofat least fifteen amino acid residues.

[0106] Analogs of the protein having the amino acid sequence shown inFIG. 8, and/or truncations thereof as described herein, may include, butare not limited to an amino acid sequence containing one or more aminoacid substitutions, insertions, and/or deletions. Amino acidsubstitutions may be of a conserved or non-conserved nature. Conservedamino acid substitutions involve replacing one or more amino acids ofthe proteins of the invention with amino acids of similar charge, size,and/or hydrophobicity characteristics. When only conserved substitutionsare made the resulting analog should be functionally equivalent.Non-conserved substitutions involve replacing one or more amino acids ofthe amino acid sequence with one or more amino acids which possessdissimilar charge, size, and/or hydrophobicity characteristics.

[0107] One or more amino acid insertions may be introduced into theamino acid sequences shown in FIG. 8. Amino acid insertions may consistof single amino acid residues or sequential amino acids ranging from 2to 15 amino acids in length. For example, amino acid insertions may beused to render the protein is no longer active. This procedure may beused in vivo to inhibit the activity of a protein of the invention.

[0108] Deletions may consist of the removal of one or more amino acids,or discrete portions from the amino acid sequence shown in FIGS. 8. Thedeleted amino acids may or may not be contiguous. The lower limit lengthof the resulting analog with a deletion mutation is about 10 aminoacids, preferably 100 amino acids.

[0109] Analogs of a protein of the invention may be prepared byintroducing mutations in the nucleotide sequence encoding the protein.Mutations in nucleotide sequences constructed for expression of analogsof a protein of the invention must preserve the reading frame of thecoding sequences. Furthermore, the mutations will preferably not createcomplementary regions that could hybridize to produce secondary mRNAstructures, such as loops or hairpins, which could adversely affecttranslation of the receptor mRNA.

[0110] Mutations may be introduced at particular loci by synthesizingoligonucleotides containing a mutant sequence, flanked by restrictionsites enabling ligation to fragments of the native sequence. Followingligation, the resulting reconstructed sequence encodes an analog havingthe desired amino acid insertion, substitution, or deletion.

[0111] Alternatively, oligonucleotide-directed site specific mutagenesisprocedures may be employed to provide an altered gene having particularcodons altered according to the substitution, deletion, or insertionrequired. Deletion or truncation of a protein of the invention may alsobe constructed by utilizing convenient restriction endonuclease sitesadjacent to the desired deletion. Subsequent to restriction, overhangsmay be filled in, and the DNA religated. Exemplary methods of making thealterations set forth above are disclosed by Sambrook et al (MolecularCloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor LaboratoryPress, 1989).

[0112] The invention also contemplates isoforms of the proteins of theinvention. An isoform contains the same number and kinds of amino acidsas a protein of the invention, but the isoform has a different molecularstructure. The isoforms contemplated by the present invention are thosehaving the same properties as a protein of the invention as describedherein.

[0113] The present invention also includes a protein of the inventionconjugated with a selected protein, or a selectable marker protein toproduce fusion proteins. Additionally, immunogenic portions of a proteinof the invention are within the scope of the invention.

[0114] The proteins of the invention (including truncations, analogs,etc.) may be prepared using recombinant DNA methods. Accordingly, thenucleic acid molecules of the present invention having a sequence whichencodes a protein of the invention may be incorporated in a known mannerinto an appropriate expression vector which ensures good expression ofthe protein. Possible expression vectors include but are not limited tocosmids, plasmids, or modified viruses (e.g. replication defectiveretroviruses, adenoviruses and adeno-associated viruses), so long as thevector is compatible with the host cell used. The expression vectors are“suitable for transformation of a host cell”, means that the expressionvectors contain a nucleic acid molecule of the invention and regulatorysequences selected on the basis of the host cells to be used forexpression, which is operatively linked to the nucleic acid molecule.Operatively linked is intended to mean that the nucleic acid is linkedto regulatory sequences in a manner which allows expression of thenucleic acid.

[0115] The invention therefore contemplates a recombinant expressionvector of the invention containing a nucleic acid molecule of theinvention, or a fragment thereof, and the necessary regulatory sequencesfor the transcription and translation of the inserted protein-sequence.Such expression vectors may be useful in the above-described therapiesusing a nucleic acid sequence encoding a OX-2 protein. Suitableregulatory sequences may be derived from a variety of sources, includingbacterial, fungal, or viral genes (For example, see the regulatorysequences described in Goeddel, Gene Expression Technology: Methods inEnzymology 185, Academic Press, San Diego, Calif. (1990). Selection ofappropriate regulatory sequences is dependent on the host cell chosen,and may be readily accomplished by one of ordinary skill in the art.Examples of such regulatory sequences include: a transcriptionalpromoter and enhancer or RNA polymerase binding sequence, a ribosomalbinding sequence, including a translation initiation signal.Additionally, depending on the host cell chosen and the vector employed,other sequences, such as an origin of replication, additional DNArestriction sites, enhancers, and sequences conferring inducibility oftranscription may be incorporated into the expression vector. It willalso be appreciated that the necessary regulatory sequences may besupplied by the native protein and/or its flanking regions.

[0116] The invention further provides a recombinant expression vectorcomprising a DNA nucleic acid molecule of the invention cloned into theexpression vector in an antisense orientation. That is, the DNA moleculeis operatively linked to a regulatory sequence in a manner which allowsfor expression, by transcription of the DNA molecule, of an RNA moleculewhich is antisense to a nucleotide sequence comprising the nucleotidesas shown in FIG. 7. Regulatory sequences operatively linked to theantisense nucleic acid can be chosen which direct the continuousexpression of the antisense RNA molecule.

[0117] The recombinant expression vectors of the invention may alsocontain a selectable marker gene which facilitates the selection of hostcells transformed or transfected with a recombinant molecule of theinvention. Examples of selectable marker genes are genes encoding aprotein such as G418 and hygromycin which confer resistance to certaindrugs, b-galactosidase, chloramphenicol acetyltransferase, or fireflyluciferase. Transcription of the selectable marker gene is monitored bychanges in the concentration of the selectable marker protein such asb-galactosidase, chloramphenicol acetyltransferase, or fireflyluciferase. If the selectable marker gene encodes a protein conferringantibiotic resistance such as neomycin resistance transformant cells canbe selected with G418. Cells that have incorporated the selectablemarker gene will survive, while the other cells die. This makes itpossible to visualize and assay for expression of recombinant expressionvectors of the invention and in particular to determine the effect of amutation on expression and phenotype. It will be appreciated thatselectable markers can be introduced on a separate vector from thenucleic acid of interest.

[0118] The recombinant expression vectors may also contain genes whichencode a fusion moiety which provides increased expression of therecombinant protein; increased solubility of the recombinant protein;and aid in the purification of a target recombinant protein by acting asa ligand in affinity purification. For example, a proteolytic cleavagesite may be added to the target recombinant protein to allow separationof the recombinant protein from the fusion moiety subsequent topurification of the fusion protein.

[0119] Recombinant expression vectors can be introduced into host cellsto produce a transformant host cell. The term “transformant host cell”is intended to include prokaryotic and eukaryotic cells which have beentransformed or transfected with a recombinant expression vector of theinvention. The terms “transformed with”, “transfected with”,“transformation” and “transfection” are intended to encompassintroduction of nucleic acid (e.g. a vector) into a cell by one of manypossible techniques known in the art. Prokaryotic cells can betransformed with nucleic acid by, for example, electroporation orcalcium-chloride mediated transformation. Nucleic acid can be introducedinto mammalian cells via conventional techniques such as calciumphosphate or calcium chloride co-precipitation, DEAE-dextran-mediatedtransfection, lipofectin, electroporation or microinjection. Suitablemethods for transforming and transfecting host cells can be found inSambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition,Cold Spring Harbor Laboratory press (1989)), and other laboratorytextbooks.

[0120] Suitable host cells include a wide variety of prokaryotic andeukaryotic host cells. For example, the proteins of the invention may beexpressed in bacterial cells such as E. coli, insect cells (usingbaculovirus), yeast cells or mammalian cells. Other suitable host cellscan be found in Goeddel, Gene Expression Technology: Methods inEnzymology 185, Academic Press, San Diego, Calif. (199 1).

[0121] The proteins of the invention may also be prepared by chemicalsynthesis using techniques well known in the chemistry of proteins suchas solid phase synthesis (Merrifield, 1964, J. Am. Chem. Assoc.85:2149-2154) or synthesis in homogenous solution (Houbenweyl, 1987,Methods of Organic Chemistry, ed. E. Wansch, Vol. 15 I and II, Thieme,Stuttgart).

[0122] The following non-limiting examples are illustrative of thepresent invention:

EXAMPLES Example 1

[0123] This example demonstrates the increased expression of certaingenes following pv immunization.

Mice

[0124] C3H/HEJ and C57BL/6 mice were purchased from The JacksonLaboratory, Bar Harbor, ME. Mice were housed five/cage and allowed foodand water ad libitum. All mice were used at 8-12 weeks of age.

Monoclonal antibodies

[0125] The following monoclonal antibodies (Mabs) from Pharmingen (SanDiego, Calif.) were used: anti-IL-2 (JES6-1A12; biotinylated JES6-5H4);anti-IL-4 (11 B11; biotinylated BVD6-24G2); anti-IFNγ (R4-6A2;biotinylated XMG1.2); anti-IL-10 (JES5-2A5; biotinylated SXC-1,Pharmingen, San Diego, Calif.); mouse IgG1 isotype control (clone 107.3,BALB/c anti-TNP). Strepavidin horse radish peroxidase and recombinantmouse GM-CSF was also purchased from Pharmingen (San Diego, Calif.).

[0126] NLDC-145 (anti-mouse dendritic cells), and F(ab′)₂ rabbitanti-rat IgG FITC conjugate (non-cross reactive with mouse IgG), orF(ab′)₂ rabbit anti-mouse IgG PE was obtained from Serotec, Canada.

[0127] Rabbit complement, L3T4, anti-thy1.2, anti-Ly2.2, anti-Ly2.1(mouse IgG3), FITC-MAC-1 and mouse IgG1 anti-rat OX-2 were obtained fromCedarlane Labs, Hornby, Ontario.

[0128] Anti-CD28 (PV-1) and anti-CTLA (UC10-4F10-11) were obtained fromDrs. C. June and J. Bluestone respectively, while anti-B7-1, anti-B7-2were obtained from Dr. G. Powers. High titres of all 4 of the latterantibodies were produced by in vitro culture in a CELLMAX system (CELLCOInc., Germantown, Md., USA).

Preparation of Cells

[0129] Spleen, Peyer's Patch (PP) and mesenteric lymph node (MLN) cellsuspensions were prepared aseptically from individual mice of thedifferent treated groups in each experiment.

[0130] Where dendritic cells were obtained by culture of bone marrowcells in vitro the following technique was used (Gorczynski et al.,1996a). Bone marrow plugs were aspirated from the femurs of donor maleC57BL/6 (or BALB/c) mice, washed and resuspended in aF10. Cells weretreated sequentially with a mixture of antibodies (L3T4, anti-thy1.2,anti-Ly2.2) and rabbit complement and dead cells removed bycentrifugation over mouse lymphopaque (Cedarlane Labs, Ontario). Cellswere washed×3 in aF10, and cultured in 10ml aF10 in tissue cultureflasks, at a concentration of 2×10⁶/ml with 500 U/ml recombinant murineGM-CSF (Pharmingen, USA). Fresh GM-CSF was added at 36 hr intervals.Cells were separated over lymphopaque on days 3.5 and 7 of culture,again reculturing in aF10 with recombinant GM-CSF. At 10 days an aliquotof the sample was stained with NLDC-145 and FITC anti-rat IgG, anti-OX-2and PE anti-mouse IgG, FITC-anti-B7-1 or FITC anti-B7-2. Mean stainingwith these antibodies using cells harvested from such cultures has been93%±7%, 14%±5%, 78%±9% and 27%±6% respectively. Remaining cells werewashed, and injected into the portal vein as described.

Portal vein immunizations and renal transplantation

[0131] The pv immunizations and renal transplantation were performed asdescribed earlier (Gorczynski et al., 1994). All C3H mice received pv/ivimmunization with 15×10⁶ C57BL/6 10-day cultured, bone marrow derived,dendritic cells, followed by C57BL/6 kidney transplantation. Animalsreceived 1 intramoscular (im) injection with 10 mg/Kg cyclosporin A onthe day of transplantation. Mice were sacrificed for tissue harvest andRNA preparation 5 days after transplantation. In other studies animalswere sacrificed as described in the text.

[0132] Where monoclonal antibodies were injected into transplanted mice,animals received 100 mg intravenous (iv) at 2 day intervals (×4injections) beginning within 2 hours of transplantation.

Cytokine Production from Spleen Cells of Transplanted Mice

[0133] In cultures used to assess induction of cytokine productionspleen responder cells stimulated with irradiated (2000R) C57BL/6 spleenstimulator cells in triplicate in aF10 have been used. In multiplestudies significant quantitative or qualitative differences in cytokineproduction from spleen, lymph node or Peyer's Patch of transplanted micehave not been seen. (Gorczynski et al., 1994b). Supernatants were pooledat 40 hr from replicate wells and assayed in triplicate in ELISA assaysfor lymphokine production. All capture antibodies, biotinylateddetection antibodies, and recombinant cytokines were obtained fromPharmingen (San Diego, Calif.-see above).

[0134] For IFNγ the assay used flat-bottomed 96-well Nunc plates (Gibco,BRL) coated with 100 ng/ml R4-6A2. Varying volumes of supernatant werebound in triplicate at 4° C., washed ×3, and biotinylated anti-IFNγ(XMG1.2) added. After washing, plates were incubated withstrepavidin-horse radish peroxidase (Cedarlane Labs), developed withappropriate substrate and OD₄₀₅ determined using an ELISA plate reader.IL-10 was assayed using a similar ELISA system with JES5-2A5 as thecapture antibody, and biotinylated SXC-1 as developing antibody. Eachassay reliably detected cytokine levels in the range 0.01 to 0.1 ng/ml.ELISA assays for IL-2 and IL-4 used JES6-1A12 and 11B11 as captureantibodies, with biotinylated JES6-5H4 or BVD6-24G2 as developingantibodies. Sensitivity of detection was 10 pg/ml for each cytokine.

Oligonucleotide Primers

[0135] The primers used for PCR amplification for b-actin, and differentcytokines, are described in previous publications (Gorczynski, R. M.,1995a; Gorczynski, R. M., 1995b; Gorczynski, R. M., 1996a). In addition,the following oligonucleotides were synthesized. cDNA (DP)5′-TTTTGTACAAGCTT₃₀-3′ (SEQ.ID.NO.:3) Adapter 1 (Ad1):5′-CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAGGT-3′ (SEQ.ID.NO.:4)Adapter 2 (Ad2): 5′-TGTAGCGTGAAGACGACAGAAAGGGCGTGGTGCGGAGGGCGGT-3′(SEQ.ID.NO.:5) PCR Primer 1 (P1): 5′-CTAATACGACTCACTATAGGGC-3′(SEQ.ID.NO.:6) Nested Primer 1 (NP1): 5′-TCGAGCGGCCGCCCGGGCAGGT-3′(SEQ.ID.NO.:7) PCR Primer 2 (P2): 5′-TGTAGCGTGAAGACGACAGAA.3′(SEQ.ID.NO.:8) Nested Primer 2 (NP2): 5′-AGGGCGTGGTGCGGAGGGCGGT-3′(SEQ.ID.NO.:9)

Driver and Tester Preparation

[0136] RNA was extracted from pooled mesenteric lymph node (MLN) andPeyer's Patches (PP) of 5/group renal transplant mice with iv or pvimmunization. Poly(A)⁺mRNA was prepared from the driver (iv) group, and2 mg material used for ds cDNA synthesis with 1 ng DP primer and a cDNASynthesis Kit (Clontech) with T4 DNA polymerase. The final cDNApreparation was digested with Rsal in a 50 ml reaction mixture with 15units enzyme (GIBCO) for 3 hrs, and the cDNA phenol-extracted, ethanolprecipitated, and resuspended in 7 ml of deionized water (concentrationapproximately 300 ng/ml).

[0137] Rsal digested ds tester cDNA (pv group) was prepared in a similarfashion. 50 ng of tester cDNA diluted in TE buffer was ligated with 2 mlof Ad1 and Ad2 (each at 10 mM) in separate ligation reactions at 16° C.for 18 hrs with 50 Units/ml T4 ligase. Thereafter 1 ml of 0.2M EDTA wasadded, the mixture heated at 70° C. for 5 min to inactivate the ligase,and the product stored at −70° C.

Subtractive Hybridization and PCR Amplification

[0138] 600 ng driver (iv) ds cDNA was added to each of two tubescontaining 20 ng Ad1- and Ad2-ligated pv cDNA. The samples were mixed,precipitated with ethanol, resuspended in hybridization buffer, overlaidwith mineral oil and denatured/annealed in standard fashion. The twoindependent samples were then combined, 200 ng fresh driver cDNA addedto allow for further enrichment of differentially expressed mRNAs, andthe mixture again denatured and annealed for 10 hrs at 68° C. The finalsample was diluted in Hepes buffer with EDTA and stored at −20° C.

[0139] After subtraction two PCR amplifications were performed on thesubtracted cDNA. In the first 1 ml of subtracted cDNA was amplifiedusing 1 ml each of P1 and P2. The conditions for amplification were asdescribed by Diatchenko. The amplified products were diluted 10-fold indeionized water and 1 ml of product used for further amplification usingthe nested primers (NP1 and NP2) and a 10-cycle amplification reaction.Aliquots of the original driver/tester and subtracted cDNAs were usedfor PCR reactions with control oligonucleotide primers (b-actin) forknown “housekeeping genes”, and with primers for genes whose expressionhas been previously documented to be different in iv/pv immunized mice.These data are shown in FIGS. 1 and 2.

[0140]FIG. 1 shows PCR validation of suppressive subtractivehybridization. Samples from unsubtracted (lanes 1, 3, 5 and 7) orsubtracted (lanes 2, 4, 6 and 8) mRNA were reverse transcribed andtested in PCR with b-actin primers for different PCR cycle times. Lanes1 and 2: 15 cycles; lanes 3 and 4: 20 cycles; lanes 5 and 6: 25 cycles;lanes 7 and 8: 30 cycles.

[0141]FIG. 2 shows PCR validation of suppressive subtractivehybridization. Samples from unsubtracted (lanes 2 and 4) or subtracted(lanes 3 and 5) mRNA were tested as in FIG. 1, except primers used werefor IL-10, and different cycle times are shown. Lanes 2 and 3: 20cycles; lanes 4 and 5: 30 cycles, lane 1: mol. wt. standard.

[0142] In addition, cloning of the subtracted cDNA was performed asfollows.

Cloning and Further Analysis of Subtracted cDNA

[0143] The PCR amplified cDNA was cloned with a TA cloning kit(Invitrogen, California) by directly ligating into the PCR II vector.Ligation was performed at an insert:vector ratio of 3:1 in 1× ligationbuffer with T4 ligase (3 U/ml) overnight at 14° C. Ligation productswere then inserted into INFaF′ competent Escherichia Coli using astandard transformation protocol, and selected with ampicillin on platescontaining X-gal (5-bromo-4-chloro-3-indolyl--D-galactoside). Miniprepplasmid DNA was purified with a Plasmid extraction Spin kit (Qiagen,Germany) and cut with EcoR I restriction enzyme to determine whether theplasmids contained the expected insert. Plasmids with inserts weresequenced by the dideoxy sequencing method using a T7 sequencing kit(Pharmacia Biotech, Canada). Nucleic acid homology searches wereperformed using the BLAST program at the National Center forBiotechnology Information (NIH, Bethesda, USA).

[0144] Further analyses of cloned material, using Northernhybridization, was as follows. Inserts in pCRII were amplified for 12cycles using nested PCR primers. The amplified material was purifiedusing Qiaquick Spin PCR Purification Kits (Qiagen), ³²P-labeled byrandom priming, and used as a probe for Northern hybridization with 20mg samples of the original (and fresh) iv or pv total RNA. Hybridizationwas performed in 5 ml of ExpressHyb solution (Clontech) with a minimumof 5×10⁶ cpm per 100 ng cDNA probe and 0.1 mg/ml sonicatedheat-denatured salmon sperm DNA. Filters were washed 4 times, each at 15min at 27° C. with 1×SSC and 0.1% SDS, followed by a high stringencywash at 42° C. for 30 min with 0.2×SSC and 0.1% SDS. Exposure timesvaried from 18 hrs to 6 days. FIG. 3 shows an autoradiograph using^(32g)P-labeled probes prepared from 4 clones obtained using thesubtraction hybridization approach described above (with pv cDNA astester material and iv cDNA as driver). A labeled control probe wasprepared with a PCR amplicon for mouse b-actin. Total RNA was preparedfrom mice receiving iv or pv immunization and equivalent amounts loadedin replicate lanes as shown, with gels developed from 18 hours (clone#28) to 6 days (#71). Clone 8 is most homologous with mouse poly (A)binding protein. Clone 16 is most homologous with rat MRC OX-2. Clone 28is most homologous with human zinc-finger protein. Clone 71 has nohomologous sequence.

Western Blotting Protocol

[0145] The technique used was essentially that described by Sandhu etal. (1991) as modified by Bronstein et al. (1992). Samples were obtained14 days post renal transplantation, using groups described in FIG. 5.Fresh rat thymus cells were used as control. Samples wereelectrophoresed in 12%SDS-PAGE and transferred to PVDF membranes (NovexCo., San Diego, Calif.) prior to addition of primary antibody. Acommercial anti-rat OX-2 was used as test reagent; control antibody wasan antibody to mouse CD8a. The developing antibody used was a commercialhorse-radish peroxidase labeled anti-mouse IgG. All reagents wereobtained from Cedarlane Labs (Hornby, Ontario, Canada).

DNA Sequence Homology Comparison

[0146] Comparison of mouse OX-2 with known cDNA sequences for B7-1,B7-2, CD28 and CTLA-4 was performed using a DNASIS program (version2.0).

Results Evaluation of Suppression Subtraction Hybridization(SSH)Technique

[0147] In order to evaluate the efficacy of the SSH technique used, theinventor used his previous evidence that, by PCR analysis, increasedexpression of mRNA for IL-10 genes was evident in lymphoid tissue frompv immunized mice. Accordingly, a dilution analysis of cDNA from thetester, driver and subtracted material, using PCR primers for b-actinand IL-10 was performed. As shown in FIG. 1, after SSH there was adetectable signal for b-actin in subtracted material only after 35cycles of amplification. By contrast, a signal was present in theunsubtracted material after only 15 cycles. Using additionalquantitative measures of template, it was found to correspond to some1000-10,000 depletion of b-actin mRNA. In a separate study, analyzingIL-10 mRNA (FIG. 2), significant enrichment of IL-10 mRNA was found asdetermined by comparison of the amplification detected at 30 cycles insubtracted/unsubtracted material (see lanes 4 and 5, FIG. 2).

[0148] In a further test of the efficiency of subtraction the mixture ofunsubtracted and subtracted tester (pv) cDNA was labeled and hybridizedto Northern blots of iv (tester) and pv (driver) total RNA. The results(data not shown) indicated that the subtracted tester cDNA probe didindeed produce a significantly stronger signal with the tester RNA.Given the evidence that for any cDNA species to produce a signal in aNorther blot it must represent a concentration greater than 0.1-0.3% ofthe cDNA mixture, these data are again consistent with our havingproduced a high level of enrichment of pv-specific cDNA, with aconcomitant reduction in abundant cDNAs common between tester (pv) anddriver (iv) material.

Detection of Unique cDNA Fragments in Tissue From pv Immunized Mice

[0149] The efficiency and validity of SSH for detection of cDNAs uniqueto the tissue sample from the pv immunized mice was further confirmedafter cloning and sequence analysis of selected tester-specific cDNAs.10 randomly selected cDNA clones (of 66 sequenced) were used to probemultiple preparations of pv or iv whole RNA. All revealed unique mRNAsexpressed preferentially in the pv samples. Autoradiograms from 4 ofthese Northern blots, along with a b-actin probe as control, are shownin FIG. 3. Exposure times from 18 hrs to 6 days were used which wereinterpreted as indicative of pv specific cDNAs of different abundance inthe samples of interest.

[0150] The cDNA inserts of the 4 clones shown, along with the other 62clones, were partially sequenced and analyzed for homology in theGenBank and EMBL data bases. A summary of these data are shown inTable 1. Note that some 30 cDNA fragments had at least 50% homology(BLAST score>250 over at least 50 nt) with other described sequences. Afurther 14 clones showed similar homology with known rat/human genes.Both sets may represent members of different gene families. Anadditional 22 clones demonstrated no significant matches with entries inthe database, and thus may represent novel genes up-regulated after pvimmunization. That the data shown are a minimal estimate of suchdifferentially expressed genes is evident from the fact that homologywith IL-4 or IL-10 gene sequences (mRNAs known to be over-expressedfollowing pv immunization-see also FIG. 2) were NOT detected in any ofthe 66 clones analyzed.

[0151] The sequence homology for the clones shown in FIG. 3 (>80%homology over the compared sequence) led to the further characterizationof these clones. Clone 8 was shown to be most homologous with mouse poly(A) binding protein; clone 16 was shown to be most homologous with ratMRC OX-2; and clone 28 was shown to be most homolgous with humanzinc-finger protein. No homologous sequence was found for clone 71. Inthe data that follows, the analysis of one of these clones which showedhomology to a rat cDNA (for OX-2, a molecule previously characterized asbeing preferentially expressed on rat thymocytes and dendritic cells) isdescribed. The rationale for further investigation of this clone lies indata showing that infusion of dendritic cells via the portal vein is apotent method for prolonging allograft survival in our model systems.Note, however, that while the bone marrow derived dendritic cells thatwere infused via the portal vein themselves express OX-2 (see above),identical data has been obtained in Northern gels to those shown in FIG.3 using tissue harvested from mice receiving, as the earlieststudies(1-5) irradiated spleen cells (OX-2⁻ by FACS analysis) via theportal vein. In addition, in both situations, OX-2 mRNA was not detectedby this suppression subtraction hybridization approach when we usedtissue harvested at 0.5-2.5 days post transplantation. These results areconsistent with the idea that the OX-2 signal detected is a result ofnovel increased expression in cells following pv immunization.

Probing a cDNA Library from Tissue from pv Immunized Mice for Expressionof the Murine Equivalent of Rat OX-2

[0152] A cDNA library was constructed from mRNA prepared from a pool of5 C3H mice receiving pv immunization with 25×10⁶ irradiated (2000 Rads)C57BL/6 bone marrow cells followed by renal transplantation as describedin the Materials and Methods, using a kit purchased from ClonTech.Clones were plated in LB medium and probed with the ^(32g)P-labeledamplicon described in FIG. 3 as showing homology with rat OX-2. A 1.3 Kbclone was detected, amplified, and shown after ³²P-labeling to detect adifferentially expressed product by Northern gel analysis. Aftersequencing using an automated DNA sequencer and fluorescent-labeleddeoxynucleotides, this 1.3 Kb fragment was found to share >95% homologywith the region encoding the 3′untranslated region of the rat OX-2 mRNAas determined from the GeneBank sequence for rat OX-2.

[0153] Using a primer construct program, a 5′PCR primer representingpositions 1-19 of the rat GeneBank sequence (corresponding to a portionof the 5′untranslated region, and the leader sequence) and 3′ primersfrom our characterized mouse sequence were synthesized, andlong-distance amplification performed to produce an amplicon predictedto encode the open-reading-frame (ORF) of the murine equivalent of therat OX-2 gene. This amplicon was determined (as expected) to be of some1.4 Kb length. Automated sequencing produced a full-length sequence forthe mouse homologue of the rat MRC OX-2 gene, including an ORF with >90%homology (predicted amino acid sequence) with the corresponding ratproduct, along with the 3′untranslated region. This sequence has beensubmitted to the Genebank (accession number AF004023).

[0154] Using a DNASIS program the predicted mouse protein sequence hassome 51% homology with B7-1 and B7-2, 48% with CD28 and 54% with CTLA4(unpublished).

Evidence for an Important Role for the Expressed OX-2 Homologue inProlonged Graft Survival following pv Immunization

[0155] In an attempt to define the potential importance of the productencoded by the OX-2 gene we used a commercial antibody to rat OX-2 in atransplant model in mice receiving pv immunization and renaltransplantation. In the first such study, it was asked whether there wasevidence for specifically increased expression of the OX-2 moleculefollowing pv immunization. By FACS analysis, using dual staining ofhepatic mononuclear cells and spleen cells with OX-2 and NLDC145,similar numbers of NLDC145⁺ cells in liver or spleen samples from iv andpv immunized mice were found, (5×10⁵ and 6.5×10⁶ respectively), but a4-fold increase in the numbers of OX-2⁺ NLDC145⁺ following pvimmunization. FIG. 4 shows a flow cytometry profile of spleen adherentcells from iv immunized/grafted mice (panels A and B) or pvimmunized/grafted mice (panels C and D). Cells were harvested 7 daysafter transplantation and stained with NLDC145 and F(ab′)₂FITC-anti-ratIgG, as well as with control (clone 107.3) mouse IgG1 serum (left handpanels) or anti-OX-2 (right hand panels) and F(ab′)₂PE-anti-mouse IgG.Data are representative of one of three different studies. Values shownrepresent the total cell population in each quadrant. The absolutenumbers (×10⁵) of double positive cells in the liver or spleen of pvimmunized mice were 3.2±0.5 and 39±8 respectively (see FIG. 4 for FACSprofiles of spleen adherent cells). This 4-fold increase was seenregardless of the cells used for pv immunization, either bone marrowderived dendritic cells (some 20% OX-2⁺-see above) or irradiated wholespleen lymphoid cells (OX-2⁻), suggesting that they were not merelydetecting surviving OX-2⁺ (donor) cells, but novel expression of OX-2 invivo.

[0156] Western blot, FIG. 5, shows increased expression of OX-2 antigenafter pv immunization. The technique used for Western blotting ispreviously described. Samples were obtained 14 days post renaltransplantation, using the groups described in FIG. 6. Fresh rat thymuscells (lane 5) were used as control. Lanes 1 and 2 represent samplespooled from 3 donors/group (iv immunized; pv immunized +infusion ofanti-OX-2 respectively). Samples in lanes 3 and 4 are from individualmice receiving pv immunization and renal transplantation only (noantibody treatment). Staining with anti-rat MRC OX-2 is shown in FIG.5B; with a control antibody (to mouse Ly2.1), anti-mouse cD8a, shown inFIG. 5A. The developing antibody used was a commercial horse-radishperoxidase labeled anti-mouse IgG. No signal was seen using the mouseIgG1 isotype control clone 107.3 (BALB/c anti-TNP)-data not shown. Dataare representative of 1 of 3 equivalent studies.

[0157] Western blotting (see FIGS. 5A and 5B) of samples prepared fromthe spleen of iv vs pv immunized and grafted mice 14 days followingrenal transplantation revealed staining of a band migrating withestimated molecular weight 43 Kd, in agreement with data elsewherereporting extensive glycosylation of this molecule in isolates from ratthymus. In mice receiving pv immunization along with in vivo treatmentwith anti-OX-2, no detectable signal was seen in Western blots (see lane2, FIG. 5). No staining was seen with a murine IgG1 isotype control(BALB/c anti-TNP, clone 107.3: unpublished), making it unlikely that theband observed was Fc receptor.

[0158]FIG. 6 is a graft showing percent survival versus days post renaltransplantation. Commercial anti-OX-2 monoclonal antibody, but notanti-mouse CD28 or anti-mouse CTLA4, reverses the graft prolongationfollowing donor-specific pv immunization. Groups of 6 C3H mice receivedC57BL/6 renal allografts with no other treatment (cyclosporin A only,-⋄-), or additional pv immunization with 15×10⁶ C57BL/6 bone marrowderived dendritic cells (−<−)as described previously. Subsets of theselatter mice received iv injection (every second day ×4 injections) with100mg/mouse of a commercial anti-rat OX-2 monoclonal antibody (--) orthe isotype control (clone 107.3, -—-), or of antibodies to mouse CD28(-•-) or CTLA4 (-*-). The animal survival for the different groups shownare pooled from 2 studies. Note that the mouse isotype control itselfproduced no modification of the increased renal graft survival followingpv immunization. * p<0.02, Mann-Whitney U-test).

[0159] In two final studies mice received pv immunization andtransplantation as before, but now also received iv injection withcommercial anti-rat OX-2 (×4 injections; 100 mg/mouse at 2 dayintervals). As shown in FIGS. 5A and B and 6 these infusions ofanti-OX-2 significantly decreased the prolonged graft survival (FIG. 6)and increased expression of OX-2 antigen (Western blotting-FIGS. 5A and5B) seen following pv immunization. No perturbation of graft survivalfollowing pv immunization was seen using additional treatments withanti-CD28/anti-CTLA4 (see FIG. 6), or, in studies not shown, usinganti-B7-1 or anti-B7-2. Again infusion of the IgG1 isotype control Mab(clone 107.3) did not alter the increased graft survival seen followingpv immunization (see FIG. 6).

[0160] In separate experiments cells were harvested from mice receivingpv immunization along with additional treatment with monoclonalantibodies as show (see Table 2). Following treatment with anti-OX-2there was no longer the altered cytokine production (with polarizationto production of IL-4 and IL-10) which the inventor has described inmultiple model systems in which animals received pv donor-specificpre-transplant immunization. Treatment with any of the other 4monoclonal antibodies tested did not produce this reversal inpolarization of cytokine production seen following pvimmunization-indeed, using these Mabs alone in the absence of pvimmunization produced a trend to increased graft survival (not shown)and significant polarization in cytokine production to increased IL-4and IL-10 production, akin to that produced by pv immunization itself(upper half of Table 2).

[0161] OX-2 is a molecule previously characterized by Barclay et al.(1981, 1982) as being preferentially expressed on rat thymocytes anddendritic cells. Dendritic cells are known to be important signallingcells for lymphocytes, which also potentially regulate cytokineproduction and graft rejection, and infusion of dendritic cells is apotent means of inducing pv tolerance. The inventor has determined thatOX-2 expression increased following pv immunization, and further studiedwhether this had any functional consequences. As shown in FIGS. 4 and 5,there is indeed significantly increased expression of OX-2 in spleencells isolated from pv immunized mice, along with the increased graftsurvival and polarization in cytokine production (FIG. 6 and Table 2).In contrast, in vivo infusion of anti-OX-2 abolishes increasedexpression of this molecule, simultaneously reversing the increasedgraft survival and altered cytokine profile seen. This data isconsistent with the possible function of OX-2⁺ cells in promotingallograft survival.

[0162] In the studies described the donor dendritic cells infused viathe portal vein were themselves OX-2⁺ (see description of materials andmethods above). However, identical data in FACS analysis (FIG. 4) andWestern Blots (FIG. 5), and from suppression subtraction hybridization(FIG. 3), have been obtained in studies in which we used irradiatedwhole spleen cells (OX-2⁻ by FACS) for pv infusion. This is consistentwith the lack of evidence for increased mRNA expression of OX-2 early(1-2 days) post transplant, as noted above. Thus it seems most likelythat an operationally important “OX-2 signal” detected in the spleen ofthe pv immunized mice can derive from new expression, rather thannecessarily from infused OX-2⁺ cells. In the absence of a polymorphicmarker for OX-2, however, it cannot be determined whether increasedexpression is from donor or host cells (or both). Indeed, it is perhapssomewhat surprising that the murine antibody to rat OX-2 cross-reacts inthe fashion shown with murine OX-2. Definitive analysis of the in vivorole of OX-2 awaits similar studies to those above, using antibodiesdeveloped against the murine OX-2 homologue-these experiments arecurrently in progress. It is also important to point out that while pvimmunization led to only a 4-fold alteration in the absolute number ofdetectable OX-2⁺ NLDC⁺ cells in the spleen/liver (see text and FIG. 4),nevertheless in the face of this 4-fold difference a clear difference inOX-2 signals in Northern gels using RNA from pv vs iv immunized mice(FIG. 3), along with evidence for a role for this quantitativedifference in the outcome of graft survival (FIG. 6) were detected.Presumably these results reflect respectively the limitation to thesensitivity of the Northern assay used, and some function of thequantitation of “co-stimulation” occurring after OX-2:OX-2 ligandinteraction.

[0163] While there was some 50% homology of the predicted proteinsequence of murine OX-2 with murine B7-1, B7-2, CD28 and CTLA4(Borriello et al., 1997), antibodies to the latter molecules did notreverse the prolonged graft survival and altered cytokine productionfollowing pv immunization (FIG. 6, Table 2—see also (Castle et al.,1993)). In fact these latter antibodies themselves, infused in theabsence of pv immunization, produced some of the same changes incytokine production induced by pv immunization (Table 2).

Example 2 Murine OX-2

[0164] This example describes the cloning and sequencing of murine MRCOX-2.

[0165] A cDNA library was constructed from MLN cells derived from adultC3H mice, preimmunized 5 days earlier with 10×10⁶ allogeneic B10.BR bonemarrow-derived dendritic cells allogeneic cells by the portal venous(pv) route, using a Cap Finder PCR cDNA library construction kit(Clontech). The inventor had previously isolated, using a PCR-SelectcDNA subtraction hybridization kit (Clontech) and RNAs obtained frompooled MLN of mice immunized by the pv route or via the lateral tailvein (iv), a 350 bp amplicon which showed over 98% homology with the3′untranslated region of rat MRC OX-2 cDNA. Northern blot analysisconfirmed that this amplicon detected a differentially expressed productin RNAs prepared from iv vs pv immunized mice. This amplicon was used toscreen 5×105 clones of the amplified library. The sequences of cDNAclones were established with an Applied Biosystems 377 AutomatedSequencer, utilizing the Dye Terminator Cycle Sequencing method (AppliedBiosystems, Foster City, Calif.). The nucleotide sequence reported inthis paper has been submitted to the GenBank/EMBL Data Bank withaccession number AF004023.

[0166] The cDNA shown in FIG. 7 has an open reading frame of 837 basepairs, and a deduced amino acid sequence (FIG. 8) of 248 amino acids, ofwhich 30 represent a cleaved leader sequence. The predicted molecularweight of this, and the equivalent molecules in rat and human, isapproximately 25 kDa. The measured molecular weight in rat thymocytes,where the molecule is highly gylcosylated, is 47 kDa.

[0167] The murine MRC OX-2 shows some 92% and 77% homology overall atthe amino acid level with equivalent molecules in rat or humanrespectively. As noted for the rat molecule, the sequence from a 203-229seems likely to represent a membrane spanning domain (highly hydrophobicregion), while the region from 229-248 is likely the intracytoplasmicregion, with a stretch of highly basic residues immediately C-terminalto position 229. Homology in the combined transmembrane and C-terminalregions with rat and human shows some 98% and 85% similarityrespectively. As predicted from membership in the Ig supergene family,there are a number of conserved Cys residues forming the disulphidebonds between b-strands of Ig-like domains, (21 and 91; 130 and 184respectively); residue 91 was previously found to be the most highlyconserved among members of the immunoglobulin superfamily. Homologybetween the N-terminal Ig-domain with rat and human, versus the nextIg-domain, is 88% and 82%, or 97% and 73% respectively. This relativeconcentration in variability between rat and mouse in the V-terminalIg-domain may be more understandable when the ligand specificity for themolecules in these species is clarified. Note that the presumedextracellular portion of the molecule (1-202) contains a number of sitesfor N-glycosylation which are preserved across species (44, 65, 73, 80,94, 127, 130 and 151). This was previously reported for the rat cDNAsequence, and inferred from the measured size of the expressed materialin rat thymocytes.

[0168] The intracytoplasmic region of the molecules has no sequenceidentity with known signaling kinases, nor does it have thewell-described consensus sequence for the immunoreceptor tyrosineactivation motif (ITAM: DXXYXXLXXXXXXXYDXL). In addition, it lackstypical SH2 or SH3 domains to serve as “docking sites” for adaptermolecules which might in turn co-opt other protein kinases in anactivation cascade. Accordingly the ligand-binding activity of theextracellular domains presumably represent the biologically importantregion of the molecule. Some possible functions attributable to ligandinteraction with OX-2 can be inferred from other data in the literature.A homologous molecule, Ng-CAM, has been reported to bind aprotein-tyrosine phosphatase via N-linked oligosaccharide residues, andprotein tyrosine phosphatases are known to play a key regulatory role inimmune responses. More recently ALCAM, another adhesion molecule memberof the Ig superfamily, the gene for which is located close to that forOX-2 on chromosome 3 in humans, has been shown to bind CD6 (a member ofthe scavenger receptor cystein rich family, SRCR), and antibodies to CD6may themselves play a role in regulating immune function.

Example 3 OX-2 Positive Cells Inhibit Type-1 Cytokine Production

[0169] The inventor has shown that hepatic mononuclear, non-parenchymal,cells (NPC) can inhibit the immune response seen when allogeneic C57BL/6dendritic cells (DC) are incubated with C3H spleen responder cells.Cells derived from these cultures transfer increased survival of C57BL/6renal allografts in C3H mice. The inventor also found that increasedexpression of OX-2 on dendritic cells was associated with inhibition ofcytokine production and renal allograft rejection. The inventor furtherexplored whether inhibition by hepatic NPC was a function of OX-2expression by these cells.

[0170] Fresh C57BL/6 spleen derived DC were cultured with C3H spleenresponder cells and other putative co-regulatory cells. The latter werederived from fresh C3H or C57BL/6 liver NPC, or from C3H or C57BL/6 micetreated for 10 days by intravenous infusion of human Flt3 ligand(Flt3L). Different populations of murine bone-marrow derived dendriticcells from cultures of bone marrow with (IL-4+GM-CSF) were also used asa source of putative regulator cells. Supernatants of all stimulatedcultures were examined for functional expression of different cytokines(IL-2, IL-4, IFNγ, TGFβ). It was found that fresh C57BL/6 splenic DCinduced IL-2 not IL-4 production. Cells from the sources indicatedinhibited IL-2 and IFNγ production, and promoted IL-4 and TGFβproduction. Inhibition was associated with increased expression of OX-2on these cells, as defined by semi-quantitative PCR and FACS analysis.By size fractionation, cells expressing OX-2 were a subpopulation ofNLDC145+ cells. This data implies a role for cells expressing OX-2 inthe regulation of induction of cytokine production by conventionalallostimulatory DC.

Materials and Methods

[0171] Mice: Male and female C3H/HEJ and B10.BR (H-2^(k/k)), B10.D2(H-2^(d/d)) and C57BL/6 (H-2^(b/b)) mice were purchased from the Jacksonlaboratories, Bar Harbour, Me. Mice were housed 5/cage and allowed foodand water ad libitum. All mice were used at 8-12 weeks of age.

[0172] Monoclonal antibodies: The following monoclonal antibodies(Mabs), all obtained from Pharmingen (San Diego, Calif., USA) unlessstated otherwise, were used: anti-IL-2 (JES6-1A12; biotinylated,JES6-5H4 ); anti-IL-4 (11B11, ATCC; biotinylated, BVD6-24G2 ); anti-IFNγ(R4-6A2, ATCC; biotinylated XMG1.2); anti-IL-10 (JES5-2A5; biotinylatedSXC-1); PE anti-B7-1/B7-2 (Cedarlane Labs, Hornby, Ontario, Canada).

[0173] Rat anti-mouse OX-2 monoclonal antibodies were prepared byImmuno-Precise Antibodies Ltd. (Victoria, BC, Canada) followingimmunization of rats with a crude membrane extract of LPS stimulatedmurine DC, followed by fusion with a non-secreting rat myeloma parentcell line (YB2/3HI.P2.G11.16Ag.20). Hybridoma supernatants were screenedin ELISA using plates pre-coated with a 40-45 Kd preparation of DCextracts run on Western gels (Barclay, A. N. 1981. Immunology 44:727;Barclay, A. N., and H. A. Ward. 1982. Eur. J. Biochem. 129:447).Positive clones were re-screened using FACS analysis of CHO cellstransduced with a cDNA clone encoding full-length murine OX-2 (Chen, Z.,H. Zeng, and R. M. Gorczynski. 1997. BBA. Mol. Basis Dis. 1362:6-10).FITC-conjugated F(ab′)2 rabbit anti-rat IgG (non cross-reactive withmouse IgG) from Serotec, Canada was used as second antibody. The Mabselected for further analysis (M3B5) was grown in bulk in a CELLMAXsystem (Cellco Inc., Germantown, Md.). A crude preparation of ratimmunoglobulin (30% saturated ammonium sulphate preparation) was used asa control Ig.

[0174] In tissue culture assays where anti-cytokine Mabs were used toconfirm the specificity of the assay used 10 mg/ml of the relevant Mabswas found to neutralize up to 5.0 ng/ml of the cytokine tested.

[0175] NLDC145 (anti-mouse DC) was also obtained from Serotec.Recombinant mouse IL-4 was a kind gift from Dr. L. Yang (The TorontoHospital); mouse rGM-CSF was purchased from Pharmingen. Recombinanthuman Flt3L (derived from CHO cells) was a kind gift from Dr. A. B.Troutt, Immunex Corp., Seattle, Wash., USA.

Renal Transplantation

[0176] Renal transplantation was performed essentially as describedelsewhere (Gorczynski, R. M. et al. 1994a. Transplantation 58:816-820).Animals were anesthetized with a combination of halothane and nitrousoxide inhalation, using novogesic for post-op analgesia. Orthotopicrenal transplantation was performed using routine procedures. In brief,Donor animals received 200 Units of heparin, and kidneys were flushedwith 2 ml of ice cold heparinized physiological saline solution, priorto removal and transplantation into recipient animals with leftnephrectomy. The graft renal artery was anastomosed to the recipient'sabdominal aorta, and the renal artery was anastomosed to the recipient'sinferior vena cava. The ureter was sewn into the recipient bladder usinga small donor bladder patch. All recipients received im injection withcefotetan (30 mg/Kg) on the day of transplantation and for 2 succeedingdays. The remaining host kidney was removed 2 days aftertransplantation, unless otherwise indicated. Treatment of recipientswith pv immunization, by monoclonal antibodies, or by oral immunizationwas as described in individual studies.

Portal Vein and Oral Immunization

[0177] Portal vein and oral immunization was performed as describedearlier (Gorczynski, R. M. 1995a. Cell. Immunol. 160:224-231;Gorczynski, R. M. et al. Transplantation 62:1592-1600). All animals wereanaesthetized with nembutal. A midline abdominal incision was made andthe viscera exposed. Cells were injected in 0.1 ml through a superiormesenteric vein using a 30 gauge needle. After injection the needle wasrapidly withdrawn and hemostasis secured without hematoma formation bygentle pressure using a 2 mm 3 gel-foam.

[0178] Bone-marrow derived dendritic cells (DC) for pv immunization wereobtained by culture of T depleted bone marrow cells in vitro with rlL-4and rGM-CSF (Gorczynski, R. M. et al. Transplantation 62:1592-1600).Staining with NLDC145 and FITC anti-rat IgG, or with FITC anti-CD3confirmed >95% NLDC145+ and <5% CD3+ cells at day 10 of culture(Gorczynski, R. M. et al. Transplantation 62:1592-1600). These cellswere washed and injected into mice or used for mixed leucocyte cultures.

Preparation of Cells

[0179] Spleen and bone marrow (Gorczynski, R. M. et al. Transplantation62:1592-1600) cell suspensions were prepared aseptically from individualmice in each experiment. Hepatic mononuclear nonparenchymal cells (NPC)were isolated essentially as described elsewhere (Gorczynski, R. M.1994b. Immunology 81:27-35). Tissue was first digested at 37° C. for 45min with a mixture of collagenase/dispase, prior to separation (15 minat 17,000 rpm at room temperature) over mouse lymphopaque (CedarlaneLabs). Mononuclear cells were resuspended in a-Minimal Essential Mediumsupplemented with 2-mercaptoethanol and 10% fetal calf serum (aF10).Where cells were obtained from Flt3L injected mice, animals were treatedby iv injection of 10 mg/mouse Flt3L daily for 10 days. After enzymedigestion recovery of liver/spleen cells from these mice was markedlyincreased compared with saline-injected controls (120×10⁶, 390×10⁶ vs7×10⁶ and 120×10⁶ respectively).

Cytotoxicity and Cytokine Assays

[0180] In cultures used to assess induction of cytotoxicity or cytokineproduction responder cells were stimulated with irradiated (2000R)stimulator cells in triplicate in aF10. Supernatants were pooled fromreplicate wells at 40 hrs for cytokine assays (below). No reproducibledifferences in cytokine levels have been detected from cultures assayedbetween 36 and 54 hrs of stimulation. In some experiments the culturesreceived 1 mCi/well (at 72 hrs) of ³HTdR and proliferation was assessedby harvesting cells 14 hrs later and counting in a well-type b-counter.

[0181] Where cytoxicity was measured cells were harvested and pooledfrom equivalent cultures at 5 days, counted, and recultured at differenteffector:target with ⁵¹Cr EL4 (H2^(b/b)) or P815 (H2^(d/d)) tumor targetcells. Supernatants were sampled at 4 hrs for assessment of specificcytotoxicity.

[0182] IL-2 and IL-4 activity were assayed by bioassay using theIL-2/IL-4 dependent cell lines, CTLL-2 and CT4.S respectively.Recombinant cytokines for standardization of assays was purchased fromGenzyme (Cambridge, Mass.). IL-2 assays were set up in the presence of11 B11 to block potential stimulation of CTLL-2 with IL-4; IL-4 assayswere set up in the presence of S4B6 to block IL-2 mediated stimulation.Both the IL-2 and IL-4 assays reproducibly detected 50 pg of recombinantlymphokine added to cultures.

[0183] In addition, IL-2, IL-4, IFNy and IL-10 were assayed using ELISAassays. For IFNγ the assay used flat-bottomed Nunc plates (Gibco, BRL)coated with 100 ng/ml R4-6A2. Varying dilutions of supernatant werebound in triplicate at 4° C., washed ×3, and biotinylated anti-IFNγ(XMG1.2) added. After washing, plates were incubated withstreptavidin-horse radish peroxidase (Cedarlane Labs, Hornby, Ontario),developed with appropriate substrate, and OD₄₀₅ determined using anELISA plate reader. Recombinant IFNγ for standardization was fromPharmingen. IL-10 was similarly assayed by ELISA, using JES5-2A5 as acapture antibody and biotinylated SXC-1 as developing antibody. rlL-10for standardization was from Pepro Tech Inc. (Rocky Hill, N.J.). Eachassay detected 0.1 ng/ml cytokine. ELISA assays for IL-2 and IL-4 usedJES6-1A12 and 11B11 as capture antibodies, with JAS6-5H4 or BVD6-24G2 asdeveloping antibodies. Sensitivity of detection was 20 pg/ml for eachcytokine. Where checked the correlation between bioassay and ELISA forIL-2 or IL-4 was excellent (r>0.90). In all studies reported below, dataare shown from ELISA assays only. Where cytokine data are pooled fromseveral studies (e.g. FIGS. 14, 16, 17), absolute values of cytokineproduction were obtained as above using commercial recombinant cytokinesto standardize the assays. In our hands, supernatants fromC3Hanti-C57BL/6 cultures, under the conditions described, reproduciblycontain 950±200 and 80±25 pg/ml IL-2 and IL-4 respectively.

Preparation of RNA

[0184] Different sources of tissue from renal-grafted female micereceiving DC and kidney allografts from male mice were harvested for RNAextraction as described elsewhere (Gorczynski, R. M. 1995a. Cell.Immunol 160:224-231). The OD280/260 of each sample was measured andreverse transcription performed using oligo (dT) primers (27-7858:Pharmacia, USA). The cDNA was diluted to a total volume of 100 ml withwater and frozen at −70° C. until use in PCR reactions with primers formurine GAPDH, B7-1, B7-2 or OX-2. The sense (S) and antisense (AS)primers were synthesized by the Biotechnology Service Centre, Hospitalfor Sick Children, Toronto, using published sequences. 5′ primers were³²P end-labeled for PCR and had comparable levels of specific activityafter purification by ethanol precipitation. 5 ml cDNA was amplified for35 cycles by PCR, and samples were analyzed in 12.5% polyacrylamide gelsfollowed by overnight (18 hrs) exposure for autoradiography. In controlstudies, using H-Y primer sets, this technique reliably detects H-Y mRNAfrom extracts of female spleen cells to which male cells are added at aconcentration of 1:105 (Gorczynski, R. M. 1995a. Cell. Immunol.160:224-231; Gorczynski, R. M. et al. Transplantation 62:1592-1600).Quantitative comparison of expression of different PCR products useddensitometric scanning of the autoradiograms. GAPDH Sense:5′TGATGACATCAAGAAGGTGGTGAAG3′ (SEQ.ID.NO.:10) GAPDH Antisense:5′TCCTTGGAGGCCATGTAGGCCAT3′ (SEQ.ID.NO.:11) B7-1 Sense:5′CCTTGCCGTTACAACTCTCC3′ (SEQ.ID.NO.:12) B7-1 Antisense:5′CGGAAGCAAAGCAGGTAATC3′ (SEQ.ID.NO.:13) B7-2 Sense:5′TCTCAGATGCTGTTTCCGTG3′ (SEQ.ID.NO.:14) B7-2 Antisense:5′GGTTCACTGAAGTTGGCGAT3′ (SEQ.ID.NO.:15) OX-2 Sense:5′GTGGAAGTGGTGACCCAGGA3′ (SEQ.ID.NO.:16) OX-2 Antisense:5′ATAGAGAGTAAGGCAAGCTG3′ (SEQ.ID.NO.:17)

Statistical Analysis

[0185] In studies with multiple groups, ANOVA was performed to comparesignificance. In some cases (as defined in individual circumstances)pairwise comparison between groups was also subsequently performed.

Results

[0186] Antigen stimulation, in the presence of hepatic NPC, inducesdevelopment of a cell population capable of inhibiting proliferation andIL-2 production on adoptive transfer:

[0187] In a previous manuscript (Gorczynski, R. M. et al.,Transplantation. 66:339-349) it was reported that C3H spleen cellsstimulated in the presence of syngeneic NPC and allogeneic (C57BL/6) DCproduced a cell population able to inhibit generation of IL-2 from freshspleen cells stimulated with C57BL/6 DC, and capable of inhibitingC57BL/6 renal allograft rejection in vivo. In order to ask whether thisfunction of NPC was MHC restricted or not, the following study wasperformed.

[0188] C57BL/6 (H2^(b/b)) spleen cells were stimulated in vitro withB10.BR (H2^(k/k)) bone-marrow derived DC, in the presence/absence of thefollowing NPC: C57BL/6; B10.BR; B10.D2 (H2^(d/d)). In addition, controlcultures were incubated with the NPC only. Proliferation and IL-2/IL-4production was measured in one aliquot of these primary cultures. Inaddition, at 5 days, cells were harvested from another set of theprimary cultures, washed, and 2×10⁵ cells added to cultures containing5×10⁶ fresh C57BL/6 spleen cells and B10.BR DC. Proliferation andcytokine production was measured in these latter cultures in standardfashion. Data pooled from three equivalent studies are shown in panelsA) and B) of FIG. 9.

[0189]FIG. 9 is a bar graph showing regulation of proliferation andcytokine production following stimulation by allogeneic DC using hepaticNPC in accordance with the methods described herein. In panel A)cultures were initiated with 5×10⁶ C57BL/6 responder spleen cells alone(group 1), or with 2×10⁵ B10.BR DC (group 2). Further groups (3-5, and6-8 respectively) contained C57BL/6 responder cells and 2×105 NPC fromeither C57BL/6, B10.D2 or B10.BR respectively (3-5) or these same NPCand B10.BR DC (6-8). Data show mean proliferation and cytokineproduction from triplicate cultures in three separate studies. In panelB) data show proliferation and cytokine production from cultures of5×10⁶ C57BL/6 responder spleen cells stimulated in triplicate with 2×105B10.BR DC alone, or with the addition also of 2×10⁵ cells harvested fromthe cultures shown in the upper panel. Again data represent arithmeticmeans of 3 separate experiments. * p<0.05 compared with control cultures(far left in each panel).

[0190] There are a number of points of interest. As previouslydocumented, addition of NPC syngeneic with spleen responder cells(C57BL/6 in this case) to cells stimulated with allogeneic (B10.BR) DCled to decreased proliferation and IL-2 production from those respondercells compared with cells stimulated by DC alone (compare groups 6 and 2of upper panel of FIG. 9, panel A). IL-4 production in contrast wasenhanced. NPC alone, whether syngeneic or allogeneic to the respondercells, produced no obvious effect (groups 3-5, panel A) of FIG. 9).Furthermore, cells from primary cultures receiving the DC+NPC mixturewere able to inhibit proliferation and IL-2 production (while promotingIL-4 production) from fresh spleen cells stimulated in secondarycultures with the same (B10.BR) DC (see panel B) of FIG. 9). However,data in this Figure make another important point. The same inhibition ofproliferation/lL-2 production in primary cultures was seen using eitherB10.BR NPC (MHC matched with the DC stimulus-group 8, panel A) of FIG.9) or with third-party B10.D2 NPC (MHC-mismatched with both spleenresponder cells and allogeneic stimulator DC-group 7, panel A) of FIG.9). Again no obvious effect was seen in cultures stimulated with B10.BRor B10.D2 NPC alone (groups 4 and 5). Finally, cells taken from primarycultures stimulated with DC and NPC from either B10.BR or B10.D2 couldalso inhibit proliferation/IL-2 production from secondary C57BL/6 spleencell cultures stimulated with B10.BR DC-again cells taken from primarycultures with NPC alone produced no such inhibition (see panel B) ofFIG. 9). Thus the inhibition of proliferation/IL-2 production andenhancement of IL-4 production seen in primary cultures, as well as theinduction of suppression measured in secondary cultures, all induced byNPC, are not MHC-restricted.

Specificity of Inhibition/Suppression Induced by Hepatic NPC

[0191] One interpretation of the data shown in FIG. 9 and elsewhere isthat NPC deliver a signal to DC-stimulated cells which is distinct fromthe antigen-signal provided by the DC themselves (and is MHCnon-restricted). This signal modulates the antigen-specific signalprovided by the DC. In order to assess the antigen-specificity of theimmunoregulation described in FIG. 9, the following experiment wasperformed.

[0192] C57BL/6 spleen responder cells were stimulated with B10.D2 orB10.BR bone marrow-derived DC, in the presence/absence of NPC fromB10.BR or B10.D2 mice. Proliferation and cytokine production wasmeasured in aliquots of these cultures as before. In addition, furtheraliquots of cells harvested from these primary cultures were added tocultures of fresh C57BL/6 spleen cells stimulated with B10.BR (panelB)-FIG. 10) or B10.D2 (panel C)-FIG. 10) DC. Again proliferation andcytokine production was measured. Data pooled from three such studiesare shown in FIG. 10.

[0193]FIG. 10 shows specificity of inhibition of proliferation ofcytokine production by hepatic NPC (see FIG. 9 and description of FIG. 9for more details). In panel A), 5×106 C57BL/6 spleen cells werestimulated in triplicate for 3 days with 2×10⁵ B10.BR or B10.D2 DC,with/without 2×10⁵ NPC derived from B10.D2 or B10.BR mice. Data shownare arithmetic means of 3 repeat studies. In panels B) and C), freshC57BL/6 responder spleen cells were cultured in triplicate with eitherB10.BR DC (panel B), or B10.D2 DC (Panel C), with/without 2×10⁵additional cells from the primary cultures (groups 1-6 in panel A).Again data represent arithmetic means of proliferation/cytokineproduction from 3 studies. * p<0.05 compared with control cultures (farleft in each panel).

[0194] Data from the primary cultures (panel A)) recapitulates theobservations made in FIG. 9, and show that NPC inhibit proliferation andIL-2 production from DC-stimulated responder cells in an antigen andMHC-unrestricted fashion. However, the data in panels B) and C) of thisfigure show clearly that adoptive transfer of inhibition using cellsfrom these primary cultures occurs in an antigen-restricted fashion,dictated by the antigen-specificity of the DC used in the primarycultures, not of the NPC used for induction of suppression. Theseauxiliary cells in the NPC population thus have a functional property ofbeing “facilitator cells for induction of suppression”. Note that inother studies (data not shown) where the final assay system involvedmeasuring cytotoxicity to allogeneic target cells, a similar inhibitionof lysis (rather than cytokine production) was seen using cellsharvested from primary cultures stimulated with DC and hepatic NPC (seeGorczynski, R. M., et al. 1998a. Transplantation. 66:000-008).

Hepatic Cell Preparations from Flt3L Treated Mice are a Potent Source ofDC and “Facilitator” Cells

[0195] It has been reported at length that pv infusion of alloantigen,or iv infusion of liver-derived allogeneic mononuclear cells inducesoperational unresponsiveness in recipient animals (Gorczynski, R. M.1995a. Cell. Immunol. 160:224-231; Gorczynski, R. M. et al.Transplantation 62:1592-1600; Gorczynski, R. M. et al. 1994a.Transplantation 58:816-820.; Gorczynski, R. M., and D. Wojcik. 1992.Immunol. Lett. 34:177-182; Gorczynski, R. M. et al. 1995b.Transplantation. 60:1337-1341). The total hepatic mononuclear cell yieldfrom normal mice is of the order of 5×10⁶ cells/mouse. In order toincrease the yield, and explore the possibility that the liver itselfmight be a source both of allostimulatory DC and “facilitator” cells 2C57BL/6 mice were exposed for 10 days to daily iv infusions of 10mg/mouse human CHO-derived Flt3L, a known growth factor for DC (Steptoe,R. J. et al. 1997. J Immunol. 159:5483-5491). Liver tissue was harvestedand pooled from these donors and mononuclear cells prepared as describedin the Materials and Methods section above (mean 130×10⁶ cells/donor).These cells were further subjected to sub-fractionation by size usingunit gravity sedimentation techniques (Miller, R. G., and R. A.Phillips. 1969. J. Cell. Comp. Physiol. 73:191-198). A typical sizeprofile for recovered cells is shown in FIG. 11 (one of 3 studies).

[0196]FIG. 11 shows OX-2 expression in a subpopulation of NPC. It is asedimentation analysis (cell profile) and FACS analysis of cellsisolated at 10 days from Flt3L-treated C57BL/6 mice. Two C57BL/67 micereceived 10 mg/mouse Flt3L iv daily for 10 days. Hepatic NPC weresedimented for 3 hrs at 4° C., and the fractions shown collected (Fxs1-4 with sedimentation velocities 2.5-3.8, 3.8-5.1, 5.1-6.4 and 6.4-8.0mm/hr respectively). Aliquots of the cells were stained in triplicatewith the Mabs shown. The remainder of the cells were used as in FIGS.12-14. Data are pooled from 3 studies.

[0197] In these same studies cells isolated from the various fractionsshown in FIG. 11 were tested as follows. Firstly, cells were stainedwith FITC-labeled Mabs to B7-1, B7-2, NLDC145 and rat anti-mouse OX-2(M3B5) with FITC anti-rat IgG as second antibody. In addition, mRNAextracted from the different cell samples were assayed by PCR forexpression of GAPDH, B7-1, B7-2 and OX-2. Data are shown in FIGS. 11(pooled from 3 separate studies) and FIG. 12 (representative PCR datafrom one experiment).

[0198]FIG. 12 shows PCR detection of B7-1, B7-2 and OX-2 in hepaticNPMC. It is a PCR analysis for mRNA expression of OX-2, B7-1 and B7-2 invarious hepatic NPC cell fractions isolated from Flt3L treated mice (seeFIG. 11). Data are representative from 1 of 3 studies.

[0199] Further aliquots of the cells were used to stimulate fresh C3Hspleen responder cells in culture. Proliferation and cytokine assayswere performed as before (see FIG. 9), and in addition cells were takenfrom these primary cultures and added to fresh secondary cultures of C3Hspleen responder cells and C57BL/6 bone marrow-derived DC. Againproliferation and cytokine production was assayed from these secondarycultures. Data pooled from 3 studies of this type are shown in FIG. 13(panels A) and B).

[0200]FIG. 13 shows that hepatic NPMC from Flt3L treated mice resultsIL-2 and IL-4 production. Stimulation of proliferation/cytokineproduction by NPC from Flt3L treated mice, and inhibition of the same(where stimulation is induced by a separate population of DC) is afunction of different cell populations. (See text and FIGS. 11-12 formore details.) Hepatic NPC fractions were derived from Flt3L treatedC57BL/6 mice and were used to stimulate C3H spleen cells in triplicatecultures, alone or in the presence of bone-marrow derived C57BL/6 DC(see panel A). Data show arithmetic means for proliferation/cytokineproduction from 3 experiments. In addition, cells harvested from theseprimary cultures were added to fresh C3H spleen cells stimulated withC57BL/6 DC (panel B), and again proliferation/cytokine productionassayed. * p<0.05 compared with control groups (far left of panel).

[0201] Finally, cells from the various fractions were infused iv into2/group C3H mice which also received C57BL/6 renal allografts as antigenchallenge. Spleen cells were harvested from these individual mice 10days after transplantation and restimulated in culture with C57BL/6 orB10.D2 DC, again with cytokines measured at 40 hrs (see FIG. 14).

[0202]FIG. 14 is a bar graph of cytokines produced from cells from C3Hmice with C57BL/b renal allografts and NPC from Flt3 treated C57BL/6donors. OX-2⁺NPC infused iv into renal transplant allograft recipientsleads to polarization of cytokine production (to IL-4, IL-10 and TGFβ)in spleen cells harvested from those mice and restimulated in vitro.Fractions of NPC from Flt3L treated C57BL/6 mice (from FIG. 11) wereinfused iv into 2/group C3H recipients, receiving C57BL/6 renalallografts (along with CsA) in standard fashion (see Materials andMethods). Mice were sacrificed 14 days after transplantation and spleencells stimulated in vitro in triplicate with C57BL/6 DC stimulatorcells. Cytokines were assayed in the supernatants of these cultures at60 hrs. Data show arithmetic means pooled from cultures in 3 studies ofthis type. * p<0.05 compared with control groups (far left-no NPCinfused).

[0203] Data in FIG. 11 show that distinct subpopulations ofslow-sedimenting cells express OX-2 in the cells harvested from Flt3Ltreated mice, when compared with cells expressing B7-1 and/or B7-2. Ingeneral expression of OX-2 and B7-2 occured in equivalentsubpopulations. Faster-sedimenting cells (Fx 3 and 4 in FIG. 11), whilestaining for NLDC145, were positive by fluorescence mainly for B7-1, notB7-2 or OX-2. Similar conclusions were reached both by FACS analysis ofcell populations (FIG. 11), and by PCR analysis of mRNA (FIG. 12).

[0204] When the functional capacity of these different cell populationswas investigated (FIGS. 13 and 14) it was found that optimal directstimulation (or proliferation and IL-2 production) was seen from B7-1expressing cells (Fxs 3 and 4 in panel A) of FIG. 13), while only OX-2expressing cells (Fxs 1 and 2 in FIGS. 11 and 12) were capable ofproducing the inhibitory effects defined earlier (FIGS. 9 & 10) in thetwo-stage culture system (panel B) in FIG. 13). These same cells (Fxs 1and 2) were in turn able, after iv infusion, to polarize cells from micegiven renal allografts to produce predominantly IL-4, IL-10 and TGFβproduction on restimulation in vitro (FIG. 14). These data areconsistent with the notion that after FltL treatment of mice expansionof a population of immunostimulatory DC occurs within the liver, whichalso contains another distinct population of (facilitator) cells whichpromote immunoregulation.

Evidence that Cell Populations with “Facilitator” Activity from theLiver of Flt3L Treated Mice Prolong Graft Survival in Vivo

[0205] Since it has been reported elsewhere that there is a goodcorrelation between treatments (such as pv immunization) which decreaseIL-2 production and increase IL-4 production from restimulated cells andprolongation of graft survival (Gorczynski, R. M., and D. Wojoik. 1994.J. Immunol. 152:2011-2019; Gorczynski, R. M. 1995a. Cell. Immunol.160:224-231; Gorczynski, R. M. et al. Transplantation 62:1592-1600), andthat increased expression of OX-2 is also independently associated withincreased graft survival after pv immunization (Gorczynski, R. M. et al.1998b. Transplantation. 65:1106-1114), the next question was whethercells isolated from Flt3L treated mice which induced inhibitory functionin vitro (see FIGS. 9, 10 and 13), and expressed increased amounts ofOX-2 (FIGS. 11, 12) were themselves capable of promoting increased graftsurvival in vivo.

[0206] Groups of 2 C57BL/6 mice received iv infusions of 10 mg/mouseFlt3L for 10 days as before. Cells were isolated from the liver byenzyme digestion, and fractionated by unit gravity sedimentation. 4pools of cells were recovered, and an aliquot stained as before in FACSwith anti-OX-2. Groups of 2 C3H mice received 10×10⁶ cells iv from the 4separate pools. A control group received saline injections only. Overthe next 48 hrs all mice received C57BL/6 renal transplants. All micereceived CsA (10 mg/Kg) on the day of renal transplantation. Data inFIG. 15 are pooled from 3 studies of this type (representing 6mice/group), and show the animal survival in these 5 different groups.

[0207]FIG. 15 shows NPC from Flt3L treated C57BL/6 mice, infused iv intorecipient C3H mice, inhibit C57BL/6 renal allograft rejection. Two micegroups received the different subpopulations of NPC derived from Flt3Ltreated mice shown in FIGS. 11 and 12. Fxs 1 and 2 were OX2⁺. Micereceived C57BL/6 renal allografts within 48 hrs along with CsA (seeMaterials and Methods). Animal survival was followed as an end point.Data shown are pooled from 3 studies (6 mice/group). *p<0.05 comparedwith mice receiving CsA only ( ).

[0208] It is quite clear from this Figure that only hepatic cellsexpressing OX-2 (Fxs 1 and 2—see FIGS. 11 and 12) were capable ofpromoting increased graft survival after iv infusion. Comparison ofthese data with those in FIG. 13 confirm that these cell populationswere also those identified, using a 2-stage culture assay system, ascells with functional “facilitator” activity (see also FIGS. 9 and 10).There was no significant difference in survival between groups receivingNPC-Fx1 or NPC-Fx2 in this experiment, in keeping with relativelyequivalent levels of OX-2 expression in these fractions (FIG. 11).

Anti-OX-2 Monoclonal Antibody in Vitro Reverses Regulation Induced byHepatic NPC

[0209] A final study was directed to whether anti-OX-2 monoclonalantibody M3B5, added to cultures of C3H spleen responder cells,allogeneic (C57BL/6) DC and NPC from C57BL/6 mice, could prevent theinhibition of IL-2 production in primary cultures, and the developmentof cells able to inhibit such cytokine responses from freshly stimulatedresponder cells in secondary cultures (see FIGS. 9, 10 and 13). Data inFIGS. 16 and 17 are pooled from 3 studies of this type.

[0210]FIG. 16 is a bar graph showing the effect of anti B7-1; B7-2; orOX-2 on primary allostimulation. It shows that anti-OX-2 Mab increasesIL-2 cytokine production in vitro after stimulation of C3H responderspleen cells with C57BL/6 DC. Subgroups of cultures contained the Mabsshown. Cytokines were assayed at 60 hrs. All data represent arithmeticmeans pooled from 3 repeat studies. *p<0.05 compared with control group(far left).

[0211]FIG. 17 is a bar graph showing that anti-OX-2 reverses inhibitionby NPC. It shows that anti-OX-2 Mab inhibits development ofimmunoregulatory cells in vitro following incubation with hepatic NPC.C3H responder spleen cells were incubated in triplicate with C57BL/6 DCalong with NPC (see FIGS. 9 and 10). Subgoups of these culturescontained the Mabs shown. Cytokines were assayed in cultures at 60 hrs(panel A). In addition, cells were harvested from all groups, washed andadded to fresh C3H responder spleen cells and C57BL/6 DC (panel B).Cytokines in these groups were assayed 60 hrs later. All data representarithmetic means pooled from 3 repeat studies. *p<0.05 compared withcontrol group from cultures of NPC with no monoclonal antibodies (farleft in Figure)-see also FIG. 16.

[0212] Primary cultures were of two types, containing C3H responderspleen cells and C57BL/6 DC alone (FIG. 16), or the same mixture withadded C57BL/6 NPC (FIG. 17). Subsets of these cultures contained inaddition either 5 mg/ml of anti-B7-1, anti-B7-2 or anti-OX-2.Supernatants from responder cells stimulated in the presence of DC onlywere assayed after 60 hrs for cytokine production (FIG. 16). For theprimary cultures incubated with both DC and NPC, supernatants wereharvested at 60 hrs and tested for cytokine production (FIG. 17A). Inaddition, cells were harvested after 5 days, washed, and added tosecondary cultures of fresh C3H responder cells with fresh C57BL/6 DC.No monoclonal antibodies were added at this second culture stage. Datafor cytokine production these secondary cultures are shown in FIG. 17B.

[0213] Addition of anti-B7-1 or anti-B7-2 to DC stimulated spleencultures led to inhibition of cytokine production (FIG. 16), while incontrast anti-OX-2 monoclonal antibody led an increase in IL-2production in these primary cultures (FIG. 16). We have reported similarfindings elsewhere (Ragheb et al-submitted for publication).Interestingly, anti-OX-2 abolished the inhibition of cytokine productioncaused by NPC in these primary cultures (FIG. 17A-see also FIGS. 9, 10and 13). In addition, anti-OX-2 prevented the functional development ofa cell population capable of transferring inhibition of cytokineproduction to freshly stimulated spleen cells (FIG. 17B).

Discussion

[0214] There is considerable theoretical as well as practical interestin understanding the mechanism(s) by which a state of antigen specifictolerance can be induced in lymphoid populations. Limits to theeffective induction of tolerance represent a major challenge to moresuccessful allo (and xeno) transplantation, to name but one example(Akatsuka, Y., C. Cerveny, and J. A. Hansen. 1996. Hum. Immunol.48:125-134). Significant efforts have been invested into exploring howpre- (or peri-) transplant donor-specific immunization might producesuch a state (Qian, J. H. et al. 1985. J Immunol. 134:3656-3663; Kenick,S., et al. 1987. Transpl. Proc. 19:478-480; Gorczynski, R. M. 1992.Immunol. Lett 33:67-77; Thelen, M., and U. Wirthmueller. 1994. Curr.Opin. Immunol. 6:106-112; Akolkar, P. N. et al. 1993. J. Immunol. 150(April 1):2761-2773; Ahvazi, B. C. et al. J. Leu. Biol. 58 (1):23-31;Albina, J. E. et al. 1991. J Immunol. 147:144-152). There is goodevidence that portal venous (pv) immunization somehow leads to toleranceinduction, and this immunoregulation can apparently be monitored byfollowing changes in cytokine production from host cells, with decreasedproduction of IL-2, IL-12 and IFNγ, and increased IL-4, IL-10, IL-13 andTGFβ (Thelen, M., and U. Wirthmueller. 1994. Curr. Opin. Immunol.6:106-112; Gorczynski, R. M. et al. 1998a. Transplantation. 66:000-008). Which, if any, of these cytokine changes is directly andcausally implicated nevertheless remains obscure.

[0215] Further analysis of the cell population able to induce toleranceafter pv immunization led to the somewhat paradoxical observation thatdonor dendritic (DC) cells represented an excellent tolerizingpopulation (Gorczynski, R. M. 1995a. Cell. Immunol. 160:224-231;Gorczynski, R. M. et al. Transplantation 62:1592-1600). Sinceantigen-pulsed DC are conventionally thought of as representing anoptimal immunizing regime, the mechanism(s) activated following DC pvimmunization which led to tolerance (Banchereau, J., and R. M. Steinman.1998. Nature. 392:245-252) was of interest. It is already clear that DCthemselves represent an extremely heterogeneous population, in terms oforigin, cell surface phenotype, turnover in vivo and possibly function(Salomon, B. et al. 1998. J. Immunol. 160:708-717; Leenen, P. J. M. etal. 1998. J. Immunol. 160:2166-2173). In the mouse lymph node at least 3discrete populations were identified, one of which comprised smallCD8a⁺NLDC145⁺cells, likely of lymphoid origin, with an immaturephenotype, and whose numbers were profoundly increased (100×) followingFlt3L treatment in vivo (Salomon, B. et al. 1998. J. Immunol.160:708-717) (administration of the latter has been reported to lead toproliferation of dendritic cells and other cells of hematopoietic origin(Maraskovsky, E. et al. 1996. J. Exptl. Med. 184:1953-1962)). Thesecells resembled the interdigitating DC found in the T cell areas of thesplenic white pulp, and have been implicated in regulation of immunityinduced by other (myeloid derived) DC (Salomon, B. et al. 1998. J.Immunol. 160:708-717; Kronin, V. et al. 1996. J. Immunol. 157:3819-3827;Suss, G., and K. Shortman. 1996. J. Exptl. Med. 183:1789-1796).

[0216] A variety of other studies have indicated that the induction ofimmunity (vs tolerance) following antigen presentation was intrinsicallydependent upon the co-existence of other signaling ligands at thesurface of DC (interacting with appropriate counter-ligands on thesurface of other cells (e.g. stimulated T cells)) (Larsen, C. P. et al.1994. J. Immunol. 152:5208-5219; Lenschow, D. J. et al. 1996. Annu. Rev.Immunol. 14:233-258; Larsen, C. P., and T. C. Pearson. 1997. Curr. Opin.Immunol. 9:641-647). It was speculated that infusion of DC via theportal vein induced tolerance by co-opting another cell population,distinguishable by expression of unique cell surface ligands, whosebiological function was to facilitate induction of tolerance, notimmunity, when antigen was presented in association with otherwiseimmunogenic DC. Some preliminary evidence supporting this hypothesis wasrecently reported (Gorczynski, R. M. et al. 1998a. Transplantation. 66:339-349). Herein, this is referred to as a facilitator cell. Moreover,because pv immunization has been shown to be associated with increasedexpression of a novel molecule, OX-2, previously reported to beexpressed on DC (Barclay, A. N. 1981. Immunology 44:727; Barclay, A. N.,and H. A. Ward. 1982. Eur. J. Biochem. 129:447; Chen, Z. et al. 1997.BBA. Mol. Basis Dis. 1362:6-10; Gorczynski, R. M. et al. 1998b.Transplantation. 65:1106-1114), it was predicted that this moleculewould in fact serve as a “marker” for the hypothetical facilitator celldescribed. Experiments reported herein are consistent with such ahypothesis.

[0217] It is here shown that within the hepatic NPC population there isa subset of cells able to inhibit stimulation by allogeneic DC in anon-MHC restricted fashion (see FIGS. 9 and 10), and able to induce thedevelopment of an antigen-specific immunoregulatory cell population invitro (see FIGS. 9 and 10). The non-MHC-restricted nature of this“facilitator” cell interaction indicates that it functions by providingan accessory signal (a regulatory not a co-stimulatory signal) to the DCwhich stimulate T cells in the allogeneic mixed leukocyte reactiondescribed, in a fashion analogous to the original description ofcostimulatory interactions (Jenkins, M. K. et al. 1988. J. Immunol.140:3324-3329). As a result the stimulated lymphocytes alter theircytokine production profile (with decreased IL-2 production andproliferation), and become able to regulate the immune response seenfrom freshly stimulated lymphocytes (see panel B in FIGS. 9 and 10).Most interestingly, following expansion of DC in vivo by Flt3Ltreatment, it is shown that in fact the liver itself contains both animmunostimulating population (large cells by velocity sedimentationanalysis), and this putative “facilitator” cell population (see FIGS.11-15). Furthermore, the latter biological activity resides within aslow-sedimenting (small size) NLDC145⁺ cell population expressingpreferentially both cell surface B7-2 and OX-2 (see FIGS. 11 and 12).When it was investigated whether this same population of cells wasactive in vivo in regulating graft tolerance, it was found again thatafter prior Flt3L treatment, the liver contained a population of cellswhich transferred increased renal graft acceptance (FIG. 15) and inparallel altered the cytokine production profile of immunized micetowards increased IL-4 and TGFβ, and decreased IL-2 and IFNy production(FIG. 14).

[0218] In a final attempt to explore the role for OX-2 expression itselfin this regulatory function, fresh spleen cells were stimulated with DCalone or in the presence of anti-B7-1, anti-B7-2 or anti-OX-2. Note thatother studies (data not shown) have confirmed that even the bone-marrowderived DC used contains small numbers of OX2⁺ cells (RMG-unpublished).Unlike anti-B7-1 and anti-B7-2 which decreased cytokine production, aresult in keeping with the hypothesized role for these as costimulatormolecules (Hancock, W. W. et al. 1996. Proc. Natl. Acad. Sci. USA.93:13967-13972; Freeman, G. J. et al. 1995. Immunity. 2:523-532;Kuchroo, V. K. et al. 1995. Cell. 80:707-718), anti-OX-2 produced asmall but significant (1.7-2.5 fold in three studies) increase in IL-2production in this system (FIG. 16). Most important, however, inclusionof anti-OX-2 Mab in a system where exogenous “facilitator” cells wereadded (from NPC), blocked completely the induction of inhibitionnormally seen in such cultures (FIGS. 9 and 10; compare with lower panelof FIG. 17). These data are consistent with the concept that OX-2delivers a regulatory, not a costimulatory, signal in this situation.

[0219] How does the present data fit within the evolving framework ofunderstanding in the heterogeneity of DC? As noted above, there has beenspeculation that a separate population of CD8a⁺NLDC145⁺ DC of lymphoidorigin which proliferates in response to Flt3L, might be responsible forimmunoregulation. Other data have implicated IL-10 as a cytokine whichmight modify development/maturation of DC into a population expressingincreased amounts of B7-2 and capable of inducing tolerance (Steinbrink,K. et al. 1997. J Immunol. 159:4772-4780). The role of regulation ofexpression of Fas as a controlling feature in this regard is unexplored(Suss, G., and K. Shortman. 1996. J. Exptl. Med. 183:1789-1796). Thedata disclosed herein is the first to implicate another molecule, OX-2,in the delivery of a tolerizing signal, perhaps in association withalterations in expression of B7-2, Fas etc. It is intriguing that whilethere is clearly a key role for intra-thymic DC in the regulation ofself-tolerance (Banchereau, J., and R. M. Steinman. 1998. Nature.392:245-252), natural expression of OX-2 was initially first describedon thymic DC (as well as within the brain) (Barclay, A. N. 1981.Immunology 44:727)-there is as yet no evidence to suggest that thisrepresents a functionally relevant expression for OX-2 in this location.However, other independent data have also implied an immunoregulatoryrole for OX-2 expression, again as assayed by altered cytokineproduction in vitro from cells stimulated in the presence/absence ofexpressed OX-2 (Borriello, F. et al. 1997. J. Immuno. 158:4548).

[0220] It has been reported that following pv immunization there is ameasurable expansion in numbers of populations of γδTCR⁺ cells capableof adoptive transfer of increased graft survival to naive recipients(Gorczynski, R. M. et al. 1996c. Immunology. 87 (3):381-389). Little isknown concerning the nature of the antigen recognized by these cells,and why, as a population, their numbers are preferentially increasedfollowing pv immunization. It is speculated that this may be explainableultimately in terms of a differential susceptibility of γδTCR⁺ vs αβTCR⁺cells to immunoregulatory signals delivered following OX-2 expression.

[0221] In conclusion, the inventor has reported for the first time thatfunctional heterogeneity in the DC pool may be understandable in termsof differential expression of OX-2 on the cell surface. Expression ofthis molecule seems to give cells the capability to induceimmunoregulation, increased renal graft survival (and altered cytokineproduction both in vivo and in vitro). The present invention suggeststhat such OX-2 expressing cells are referred to as “facilitator” cells(for tolerance induction).

Example 4 Preparation of Murine Antibodies

[0222] Mouse and rat hybridomas to a 43 Kd molecule expressed in thethymus, on a subpopulation of dendritic cells, and in the brain, inmammalian tissue derived from mouse, rat and human were prepared. UsingCHO cells transiently transfected with adenovirus vector(s) expressing acDNA construct for the relevant OX-2 gene, the monoclonal antibodies(Mabs) detect a molecule encoded by this construct (rat OX-2 (rOX-2),mouse OX-2 (mOX-2) and human OX-2 (huOX-2) respectively). Furthermore,at least some of the anti-rat Mabs detect determinants expressed on themurine OX-2 molecule.

Materials and Methods

[0223] Antigen preparation from tissues and Western blotting wereperformed as described in Gorczynski et al., Transplantation, 1998,65:1106-1114:

[0224] Spleen cells (human samples were obtained from cadavers at thetime of organ retrieval for transplantation) were used for preparationof dendritic cells/macrophages. Tissue was digested with a mixture ofcollagenase and dispase and centrifuged over lymphopaque. Cells wereadhered for 2 hr at 37° C., washed vigorously, and incubated for 14 hrat 37° C. Dendritic cells were isolated as non-adherent cells(Gorczynski et al., Transplantation, 1996. 62:1592-1600). Routinestaining of mouse splenocytes with NLDC-145 and FITC anti-rat IgG, orFITC-MAC-1 before and after overnight incubation produced the followingstaining pattern in these adherent cells: 8%±2%, 90%±11% and 92%±9%,9%±3% respectively. The crude (non-adherent) dendritic cell preparationwas extracted with lysis buffer, titred to a protein concentration of 10mg/ml, and used for immunization. Some of the same material was usedsubsequently in screening ELISAs (below).

[0225] When brain tissue was used in Western gel analysis, whole tissueextract was electrophoresed in 12%SDS-PAGE and transferred to PVDFmembranes (Novex Co., San Diego, Calif.). Putative anti-OX-2 Mabs wereused as test reagent, with isotypic antibodies (negative in ELISA tests)used as controls. Membranes were developed using either anti-rat oranti-mouse horse radish peroxidase and appropriate substrate.

Immunization and Production of Mabs

[0226] Four female BALB/c mice were initially immunized byintraperitoneal injections with 1 mg of human or rat dendritic antigenin Complete Freundis Adjuvant. Three subsequent boosts were administeredas above, spaced at 3 week intervals, with Incomplete Freundis Adjuvant.When the serum titre had risen more than 10-fold from a pre-immune serumsample, as determined by ELISA, the 2 highest responders were boostedintravenously. Three days later the donor mice were sacrificed and thespleen cells were harvested and pooled. Fusion of the splenocytes withX63-Ag8.6.5.3 BALB/c parental myeloma cells was performed as previouslydescribed (Kohler, G. and C. Milstein. 1975. Nature. 25: p. 256-259),except that one-step selection and cloning of the hybridomas wasperformed in 0.8% methylcellulose medium (Immuno-Precise AntibodiesLtd., Victoria, BC). This proprietary semi-solid medium allows HATselection and cloning in a single step and eliminates the overgrowth ofslower growing desirable clones by faster growing, perhaps undesirable,hybridomas. Clones were picked and resuspended in wells of 96-welltissue culture plates in 200 ml of D-MEM medium containing 1%hypoxanthine/thymidine, 20% Fetal Bovine serum, 1% OPI, and 1×10⁶/mlBALB/c thymocytes. After 4 days, the supernatants were screened by ELISAfor antibody activity on plates coated with the immunizing antigen.Putative positive hybridomas were re-cloned by limited dilution cloningto ensure monoclonality and screened in FACS on extracts prepared frombrain tissue (below).

[0227] For the production of rat mAbs, 2 Fisher rats were immunized asabove with mouse antigen. Essentially the same procedure was followed,except the parental cell line used for the fusion was YB2/0.

ELISA and FACS Analysis of Putative Mabs

[0228] ELISA assays used polystyrene plates pre-coated with 10 ng/mlpoly-L-lysine, followed by overnight incubation with the crude dendriticcell antigen (used for immunization) at 10 mg/ml. Wells were developedafter binding of hybridoma supernatants using the anti-rat/anti-mousehorse radish peroxidase antibodies above and plates were analysed in anautomatic ELISA plate reader (TiterTek Multiskan, MCC/340, FlowLabs,Mississauga, Ontario, Canada).

[0229] FACS analysis was performed using putative anti-OX-2 Mabs and thefollowing cells. Fresh peripheral blood leucocytes (PBL), isolated overrat/mouse lymphopaque (Cedarlane laboratories) or Ficoll-Hypaque(human); fresh spleen dendritic cells (isolated after adherence andovernight incubation, as above); and CHO cells transduced with viralvectors engineered to contain a single copy of a cDNA inserted into thenot1/bamH1 sites, encoding the relevant species-specific OX-2, as perpublished sequences (Chen, Z. et al. 1997. BBA. Mol. Basis Dis.1362:6-10; McCaughan, G. W., et al. 1987. Immunogenetics. 25: p.133-135), or with control vector alone. FITC anti-mouse (or anti-rat)IgG was used as secondary antibody.

Mixed Leucocyte Reactivity (MLR) and Cytokine Production

[0230] Allogeneic MLR cultures, using 1:1 mixtures of 2.5×10⁶ responderPBL and mitomycin C treated stimulator PBL, were set up in 24-wellculture plates in 1 ml of aMEM medium supplemented with 10% FCS. Cellswere obtained from C3H responder mice (with stimulator C57BL/6), Lewis(LEW) rats (with Brown Norway, BN, as stimulator), and individual humandonors. Culture supernatants were harvested at 60 hrs and tested fordifferent cytokines using previously described ELISA assays (mouse), orusing CTLL-2 as bioassay for IL-2 production from all responder cellsources (Gorczynski, R. M., et al. 1998c. Immunology. 93: p. 221-229).

Results Evaluation of a Number of Mabs for Staining of Cell Populationsin Fresh PBL or Spleen

[0231] All Mabs tested in the experiments herein described werepreviously screened as described in the Materials and Methods above, anddetected a molecule in Western gel of brain extracts with MolecularWeight 42-45 Kd, and also stained CHO transduced by OX-2 encoding viralvectors. Data in Table 3 show FACS analysis for these Mabs using freshcells. The data are summed over several independent analyses, using anumber of Mabs directed to rat, mouse or human OX-2, for staining ofcells harvested from fresh PBL or spleen (adherent cells only weretested for the latter: these represented some 5%-8% of the total cellpopulation in all cases).

[0232] It is clear from Table 3 that PBL in all species tested containedsome 1.3%-2.5% OX2⁺ cells by FACS analysis, and that spleen adherentcells similarly contained 4%-8% OX2⁺ cells. As confirmation of theinventor's previous work, spleen adherent cells taken from C3H mice orLEW rats treated 4 days earlier by portal venous immunization with20×106 (or 50×10⁶ respectively) of C57BL/6 (or BN) bone marrow cellsshowed some 3.5-5 fold elevation in OX2⁺ cells (see Table 3). Underthese conditions specific increases in survival of subsequentallo-transplanted cells/tissue have been reported (Gorczynski, R. M. etal. 1996a. Transplantation 62:1592-1600).

Ability of Anti-OX-2 Mabs to Modulate Cytokine Production in MLR inVitro

[0233] In a final study the issue of whether these Mabs can modify theimmune response (as assayed by cytokine production) of cells stimulatedin an allogeneic mixed leucocyte reaction (MLR) in vitro was addressed.The inventor has previously shown that cells taken from mice pretreatedby portal allogeneic immunization produce predominantly type-2cytokines, and that an anti-OX-2 Mab could apparently reverse thispolarization in cytokine production (and indeed abolish the increasedgraft survival seen in such mice). Data in Table 4 confirm these resultsusing 3 independent Mabs to mouse OX-2. Further, rat or human cellsstimulated in the presence of anti-rat (or human) OX-2, similarly showmore pronounced IL-2 production than cells stimulated in the presence ofisotypic control Ig (or no Ig), without a generalized increase incytokine production (as analysed here by no change in IL-6 production inany group).

Discussion

[0234] In the data in this example it is confirmed that using speciesspecific Mabs, to human, rat or mouse OX-2, that Mabs to the moleculedetected on the surface of host dendritic cells may play a role inregulating cytokine production after allostimulation in vitro, and moreparticularly that functionally blocking OX-2 expression leads toenhanced IL-2 production (a type-1 cytokine) after allostimulation(Table 4). Borriello et al also recently reported that OX-2 expressionwas not a costimulator for induction of IL-2 and IFNγ synthesis(Borriello, F. et al. 1997. J. Immuno. 158:4548)-our data imply it is infact a negative signal for type-1 cytokine production. In micepreimmunized by the portal vein, as reported earlier, there is a 4-foldincrease in OX-2 expressing cells in PBL and spleen, and a reversal ofpolarization in cytokine production (from type-2 cytokines to type-1cytokines) after stimulation of cells in the presence of OX-2 (seeTables 3 and 4) (Gorczynski, R. M. et al. 1998b. Transplantation.65:1106-1114).

Example 5 Preparation of Rat Antibodies

[0235] Five rats were immunized using GERBU adjuvant (GERBU Biotechnik,Gaiberg, Germany) with 500 mg of membrane protein purified from themouse dendritic cell (DC) line DC2.4 (a gift from K. Rock, Harvard).Serum from these rats was tested 7 days after the third immunization,and compared with a pre-immunization sample in an ELISA usingplate-bound material of Mol. Wt. 40 Kd-45 Kd eluted from Western blots,and Alk Pase anti-rat Ig. Two rats with high titre antibody werere-immunized and sacrificed 4 days later for fusion of spleen cells withHAT-sensitive Sp2/0 parent cells for preparation of hybridomas.Hybridomas were screened by ELISA (56/960+ve), subcloned, and frozen(−70° C.). For further specificity testing of the anti-OX-2 Mabs willuse CHO cells can be transfected with a pBK eukaryotic expression vector(Stratagene, Calif.) expressing OX-2. Full length OX-2 cDNA, includingthe leader sequence, was amplified from DC2.4 cells using sense andantisense primers constructed with Spe1 or Xba1 sites respectively attheir 5′ ends for directional cloning into the vector. A band of theexpected size (849bp) was obtained on agarose gel electrophoresis. Thesequence of the cloned cDNA was confirmed by sequencing using anautomated DNA sequencer (Chen, Z. and Gorczynski, R. M. 1997. Biochem.Biophys. Acta. 100, in press). CHO cells were transfected byelectroporation (5×106 cells in 0.5 ml were pulsed at 960MH₂ and 120Vusing a Bio-Rad Gene Pulser (Bio-Rad, Hercules, Calif.), using the fulllength OX-2 expression plasmid along with a plasmid encoding puromycinresistance (100:1 ratio), followed by selection in puromycin (12 mg/mlfor 4 days). Puromycin resistant cells were cloned by limiting dilution.5 CHO transfectant clones have been obtained expressing mRNA for OX-2 asconfirmed by PCR. These clones can be used to screen the putative ratanti-mouse OX-2 Mabs.

[0236] (a) FACS Staining of Cells from pv Immunized Mice with Anti-mouseOX-2

[0237] A 4-fold increase in staining of spleen and hepatic NLDC145+(dendritic cell marker) cells from pv immunized mice with anti-ratOX-290 was observed. Spleen and liver tissue of mice at various times(12 hours; 2, 7 and 14 days) following pv immunization can be sectionedand stained by immunohistochemistry, using anti-NLDC145, anti-OX-2 Mabs.Single cell suspensions from the same tissues can be stained, using3-color FACS, with FITC-anti-mouse OX-2, rhodamine-anti-NLDC145, andphycoerythrin-anti-T200 (mouse lymphocyte marker). In all cases (bothFACS and immunohistochemistry) the appropriate irrelevant isotypecontrol antibodies are included. Tissue from control mice receivingrenal grafts alone, or following additional iv immunization, can also beexamined. Detection of NLDC145+ (and/or MAC-1+) cells showing increasedexpression of OX-2 is predicted in pv immunized mice only (seeGorczynski, R. M. et al. 1998. J. Immunol. 160, in press). The inventorhas shown DC-associated antigen persists only in animals with survivinggrafts (Gorczynski, R. M., Chen, Z., Zeng, H. and Fu, X. M. 1998.Transplantation submitted). It was also assessed whether anti-OX-2,infused at different times post transplantation, causes rejection (b).

[0238] (b) Modulation of Graft Rejection and Cytokine Production byAnti-mouse OX-2

[0239] C3H mice receive pv immunization with cultured C57BL/6bone-marrow derived dendritic cells (DC), CsA and renal allografts.Groups of mice receive intravenous infusion of various rat anti-mouseOX-2 Mabs (100-500 mg/mouse, ×5, at 2 day intervals), beginning atdifferent times post transplantation (this will be guided by data from(a)). Serum creatinine and animal survival are followed. Serum fromMab-treated mice are tested in ELISA and by FACS with OX-2 expressingCHO transfectants (above) to ensure antibody excess. If OX-2 expressionis important for pv induced increased graft survival, the anti-OX-2treated pv immunized mice will reject grafts like untreated controls,with similar polarization of cytokine production to type-1 cytokines(assayed by PCR; ELISA with cultured, restimulated cells). As controlspv immunized, grafted mice receive anti-CD28 and anti-CTLA4 these Mabsdo not modify the effects of pv immunization as assayed by graftsurvival or polarization in cytokine production. It is expected thatOX-2 treatment but not other Mabs, will simultaneously abolish expansionof γδTCR+ cells after pv immunization.

Example 6 Preparation of a Fusion Protein Linking the ExtracellularDomain of OX-2 to Mouse Fc

[0240] Immunoadhesins, in which a hybrid molecule is created at the cDNAlevel by fusing the extracellular domain (ED) of an adhesion moleculewith the carboxyl terminus of IgG heavy chain, the whole being expressedin mammalian cells or in a baculovirus system, have been powerful toolsin the identification and isolation of the counter ligands for theadhesion molecule of interest. Ligands for a number of members of theTNFR family, were identified in this fashion (Goodwin, R. G. et al.1993. Eur. J. Immunol. 23, 2631-2641; Gruss, H. and Dower, S. 1995.Blood 85, 3378-3404). Interest has developed in the potentialapplication of immunoadhesins as therapeutic agents. A CTLA4immunoadhesion, with the capacity to bind both B7-1 and B7-2, has beenused to inhibit T cell costimulation and decrease rejection (Larsen, C.P. et al. 1996. Nature 381, 434-438). Note that CD28/CTLA4 are notcounter ligands for OX-289. The fusion protein, is predicted to altercytokine production (increased IL-4, IL-10; decreased IL-2, IFNγ) andincrease renal graft survival like pv immunization. We expect thatsynergistic blockade of costimulation (e.g. by CTLA4-Fc) and triggeringof a coregulatory pathway (by OX-2ED-Fc) will induce tolerance andproduce indefinite graft survival.

[0241] a) Construction of an OX-2 Fusion Protein with Murine IgGFc2a

[0242] A cDNA encoding the extracellular region of OX-2 (OX-2ED) wasamplified by PCR, using a 5′ oligonucleotide primer which inserts a Sal1site 5′ immediately at the start of the V-region sequence and a 3′primer which creates a BamH1 site at the 3′ end (the site of junctionwith Fc). Using cDNA prepared from mouse ConA activated spleen cells,with a 5′primer containing an Spe1 site, and a 3′ primer containing aSal1 site, the signal peptide for IL-6 (SP-IL-6) was amplified by PCRand ligated to the OX-2ED amplicon. In frame ligation across thejunction of SP-IL-6 and OX-2ED was checked by manual sequencing-thefinal cDNA amplified by the 5′SP-IL-6 primer and the 3′OX-2ED primerwas, as expected, 695bp. A plasmid expressing murine IgGFc2a (Fcg2a),modified to create a unique BamH1 site spanning the first codon of thehinge region, and with a unique Xba1 site 3′ to the termination codon,has been obtained from Dr. Terry Strom (Zheng, X. X. et al. 1995.Journal of Immunology. 154, 5590-5600). The IgGFc2a in this insert hasbeen further modified to replace the C1q binding motif (rendering itnon-lytic) and inactivate the FcgR1 binding site (see Zheng, X. X. etal. 1995. Journal of Immunology. 154, 5590-5600). Ligation of OX-2ED andIgGFc2a in the correct reading frame at the BamH1 site yields a 1446bplong open reading frame encoding a single 478-amino acid polypeptide(including the 24-amino acid IL-6 signal peptide). The homodimer has apredicted 105 kDa Mol Wt, exclusive of glycosylation. The fusion gene isthen cloned as an Spe1-Xba1 cassette into the eukaryotic expressionplasmid pBK/CMV (Stratagene, Calif.). This plasmid has a CMVpromoter/enhancer and a neomycin-resistance gene for selection usingG418. The appropriate genetic construction of the OX-2ED-Fc can beconfirmed by direct sequencing after cloning into the plasmid vector(Chen, Z. and Gorczynski, R. M. 1997. Biochem. Biophys. Acta. 100, inpress)-see also above. The plasmid is transfected into CHO cells byelectroporation (see above), and selected in medium with 1.5 mg/ml G418(Geneticin:Life Technologies, Inc.). After subcloning, high producingclones are selected by screening culture supernatants in ELISA usinganti-OX-2 Mabs as capture antibody, and Alk Pase coupled anti-IgGFc2a asdetection antibody. OX-2ED-Fc fusion protein is purified from culturesupernatants using protein A-Sepharose affinity chromatography, dialysedagainst PBS, filter-sterilized and stored in aliquots at −20° C. Thesize, and OX-2 (+IgGFc2a) specificity of the secreted product can beconfirmed using Western blot analysis under reducing (+DTT) andnon-reducing (−DTT) conditions, with Mabs to OX-2 and rat monoclonalanti-mouse IgGFc2a (Pharmingen). The product can be titrated as aninhibitor for FACS staining of OX-2 expressing CHO cells (see above)using rat Mabs to OX-2 as probe. As a prelude to studies (below) usingOX-2ED-Fc in vivo, the half-life (t1/2) in mouse serum followinginjection of groups of 6 8-week C3H mice will be studied. This iscarried out by subjecting mice to iv injections of 50 mg or 10 mg ofOX-2ED-Fc, and obtains serial 50 ml blood samples at 0.3, 1, 6, 24, 48,72 and 96 hours. The serum is analyzed in ELISA using plates coated withanti-OX-2 as capture antibody, and Alk Pase coupled monoclonalanti-IgGFc2a for detection (thus ensuring the assay detects onlyOX-2ED-Fc, not OX-2 or IgGFc2a alone). Based on earlier data in which Fcfusion proteins were used to extend the in vivo half-life, a t1/2 in therange of 30-40 hrs (Zheng, X. X. et al. 1995. Journal of Immunology.154, 5590-5600) is predicted.

[0243] b) OX-2: IgGFc Immunoadhesion Inhibits MLR

[0244] CHO cells were transduced with a vector carry the OX-2:Fc cDNAinsert. Supernatant was harvested from the CHO cells at 7 days and wascultured with 5×10⁶ LEW spleen and 2.5×10⁶ irradiated LBNFI spleencells. The supernatant contained 50 ng/ml OX-2:Fc.

[0245] The results, shown in Table 5, demonstrate that the solubleOX-2:Fc immunoadhesion inhibits IL-2 production and generation ofcytotoxic T cells and induces IL-4 production. These results support theuse of OX-2 as an immunosuppressant.

[0246] c) Use of OX-2:Fc in Vivo for Prevention of Graft Rejection

[0247] It was shown in (b) that incubation in the presence of 50 ng/mlOX-2:Fc can inhibit an in vitro MLR reaction. To detect inhibition of invivo graft rejection, C3H mice received C57BL/6 skin grafts along withiv injection of OX-2:Fc (50 mg/mouse) every 2 days ×4 injections. Graftswere inspected daily after 10 days for rejection. In a separate study 3mice/group (receiving saline or OX-2:Fc) were sacrificed at 10 days andspleen cells restimulated in vitro (×48 hrs) for analysis of cytokineproduction. Data for these studies is shown in Tables 6 and 7. It isclear from these data that OX-2:Fc has the potential for use as animmunosuppressant to prolong graft acceptance. Furthermore, inassociation with increased graft survival in this model, OX-2:Fc alterspolarization in cytokine production, as already described for portalvein donor-specific immunization.

Example 7 OX-2 Expression in Placenta

[0248] Using in situ hybridization, the inventor has shown that OX-2 isnot expressed in the placenta of mice with increased potential for fetalloss. In contrast, OX-2 is expressed in the placenta of normal,non-aborting mice.

[0249] CBA/J and DBA/2J mice were used. Matings of CBA/J(females) withDBA/2J males show a high incidence of fetal loss (>80%), unlike thereverse scenario. Placental tissue was obtained from matings at 8.5 daysof gestation. Uteri were snap frozen, 5 mm sections cut, and stainedwith a biotinylated anti-sense probe for murine OX-2. Data shown inFIGS. 18A and 18B indicate increased expression of OX-2 mRNA (in situlabeling) in the non-aborting strain combination, with essentiallyabsent expression in the aborting combination. These data are consistentwith the notion that OX-2 expression prevents spontaneous fetal losssyndrome.

[0250] The data show that there are fewer OX2⁺ implantation sites on day8.5 of pregnancy in mice which are predisposed to fetal loss syndrome(CBAxDBA/2 matings) by contrast to CBAxBALB/c matings which are not sopredisposed. Fgl2 is the trigger for loss, and where OX-2 is alsoexpressed, these potentially doomed implantations are “rescued”. Thisfollows from the finding that the abortion rate is lower than expectedfrom % fgl2++ implantation sites, unless anti-OX-2 mAb is administered.In the latter instance, the abortion rate rises to equate with theestimated proportion of flg2++ implant sites.

Example 8 OX-2 Prevents Fetal Loss

[0251] Successful pregnancy in allopregnant mice can also be viewed asdependent upon control of graft rejection. Proinflammatory Th1 cytokines(TNF-α+IFN-γ+IL-1) can cause spontaneous abortion in mice by a mechanismwhich involves a novel prothrombinase, fgl2, which promotes fibrindeposition. However, the inventors found that spontaneous abortion ratesin abortion-prone CBA x DBA/2 matings and in low abortion rate CBA xBALB/c matings were lower than the frequency of implantation sitesshowing fibrin^(hi)+fgl2 mRNA^(hi). OX2 expression was present in thesame sites as fgl2 mRNA, and neutralization of this OX2 expression byanti-OX-2 raised the abortion rate to predicted levels. Conversely, anOX2 immunoadhesin dramatically reduced the abortion rate. Therefore, inaddition to its role in organ and tissue allograft rejection, OX2expression is involved in the prevention of spontaneous abortiontriggered by cytokine up-regulation of fgl2 at the feto-maternalinterface.

[0252] In the Example detailed below, evidence is shown using anti-OX2monoclonal antibody and a soluble form of OX2 in which the extracellulardomains of the molecule are linked to an Ig Fc region (OX2:Fc)(described in Example 6), that OX2 is fundamentally important toachieving successful allopregnancy.

Methods

[0253] All of the techniques used, including mixed leukocyte cultures,cytokine analysis, and allografting, are detailed in previouspublications (Gorczynski et al. 1996a; Arck et al. 1997a; and Clark1999). The anti-OX2 mAb (3B6) was obtained from BioCan (Mississauga,Ontario) (Gorczynski et al. 1998b). 100 μg/mouse was used for eachinjection. A polyclonal, affinity-purified, rabbit antibody to fgl2 wasdescribed elsewhere, and used ip at a dose of 22 mg/mouse. OX2Fcimmunoadhesin was given ip (35 μg/mouse).

Results In Situ Expression of OX2 mRNA Following Renal Transplantationor Allopregnancy

[0254] OX-2 has been reported at the fetomaternal interface usingimmunohistochemistry in rats (Example 7 and Bukovsky et al. 1984). Todetermine whether OX-2 was expressed in the uterus of allopregnant mice,the inventors carried out in situ hybridization for OX-2 mRNA in CBA xDBA/2 and CBA x BALB/c matings. Adjacent sections of the tissue sampleswere also used to stain for fgl2 mRNA (fgl2 is a prothrombinase moleculeup-regulated by certain Th1 cytokines implicated triggering pregnancyloss). For comparison, we also examined OX2 expression in liver sectionsfrom C3H mice receiving C57BL/6 renal allografts followingdonor-specific portal vein preimmunization, a treatment which promotestolerance and which is critically dependent upon up-regulation ofexpression of OX-2 on hepatic APC. Typical patterns for uterine staining(pregnant mice) are reported elsewhere (Clark et al. 2001), along withcumulative data for OX2 and fgl2 expression in the uteri of pregnantcontrol mice and mice treated with TNFα+IFNγ to increase abortion rates(Clark et al. 2001).

[0255] In these studies the inventors found that OX2 mRNA expression wasup-regulated following pv immunization and renal transplantation, and inallopregnant mice. In pregnant mice the inventors found a negativecorrelation between expression of the molecules fgl2 and OX-2 which didnot reach statistical significance (Clark et al. 2001). With followingcytokine treatment of pregnant animals, and prior to the onset ofabortions, the proportion of fgl2^(hi) implantations increased, althoughthis also did not achieve significance due to small numbers. However,with cytokine treatment, the proportion of OX-2^(hi) implants decreaseddramatically. These data support the hypothesis that in pregnancy, fgl2and OX-2 expression are reciprocally regulated by cytokines, that theirlevels affect pregnancy outcome, and that a major determinant of successor failure of fgl2^(hi) ‘at risk’ implantations was the presence orabsence of OX2. Most interestingly, the inventors also reported thatcontinued expression of OX2, as occurs in pregnancy, was essential forsuccessful survival of allografts following pv pretransplantimmunization, and for the concomitant changes in cytokine productionseen in those animals (previous Examples and Gorczynski et al. 2000).

Effect of Anti-OX2 mAb on Renal Transplant Survival and PregnancyOutcome

[0256] Fifty to 70% of implantations in control and cytokine-boosted CBAx DBA/2 pregnancies show the fgl2^(hi) phenotype (Clark et al. 2001). Aninjection of anti-Vg1.1 on day 8.5 of pregnancy, 1 day before abortionsbecome evident, inactivates most of the trophoblast-recognizing γδsubset producing IL-10 and TGFβ and boosts abortion rates toapproximately 48% (Arck et al. 1999). The inventors hypothesized thatthe “suppressor” γδT cells inactivated by this treatment might bedependent upon OX2 expression for their functional activity. Theinventors have reported that following allotransplantation, the kineticsof expression of OX2 follows closely the development of immunoregulatoryγδT cells. Functional blockade of OX2 expression (by anti-OX2 treatment)reverses increased graft survival (see FIG. 2) and prevents adoptivetransfer of tolerance by γδT cells. To test whether OX-2 expression inpregnancy might similarly be activating anti-abortion mechanisms which‘rescued’ fgl2^(hi) implant sites from proceeding to embryo death whereboth OX-2 and fgl-2 were expressed, the inventors injected control CBA xDBA/2- and CBA x BALB/c-mated mice with the same anti-OX-2 monoclonalantibody that blocks induction of transplantation tolerance. FIG. 19shows that injection on or after day 8.5 increased the spontaneousabortion rate to that expected if all fgl2^(hi) sites in CBA x DBA/2proceeded to resorb. The increase in abortion rate was not due to atoxic effect of anti-OX-2 on the embryo because co-administration ofanti-fgl2 to neutralize prothrombinase activity abrogated the boost inabortion rates (data not shown). Injection of anti-OX-2 into CBA xBALB/c-mated mice also increased the abortion rate to 22%, consistentwith the 21% fgl2 mRNA^(hi) implantation site frequency.

Infusion of OX2 Immunoadhesin Modulates Renal Allograft Rejection andSpontaneous Abortion

[0257] As further “proof of principle” that OX2 expression isfunctionally important for increased allograft survival the inventorsused an immunoadhesin (OX2:Fc), in which the extracellular domains ofOX2 were linked to a murine IgGFc region, to investigate modulation ofallograft rejection and pregnancy. Data in the previous Examples hasalready indicated that this molecule has potent immunoregulatoryproperties in vivo, including the ability to decrease allograftrejection. Data in FIG. 20 represent a cumulative comparison of theeffect of infusion of OX-2:Fc on renal allograft survival or rate ofabortions in CBA x DBA/2 mated mice. Once again there was a clearparallel between the functional activity of OX2:Fc measured in these twoassays.

Discussion

[0258] The notion that the rejection of organ allografts would bemimicked immunologically by immune recognition of the fetus inallopregnant mothers has been with us for decades. There has beenintense interest in the role of altered cells and soluble factors (e.g.cytokines) in the phenomena seen in both situations. As an example, withdecreased graft rejection (and successful pregnancy) there are numerousreports of the presence of unique γδT cells with “suppressorphenotypes”, and altered cytokine patterns, with elevated levels oftype-2 cytokines, in particular IL-10 and TGFβ (Chaouat et al. 1999;Tsuda et al. 2001; Clark et al. 2001b; Arck et al. 1999 and Arck et al.1997a). In contrast, allograft rejection in rodents and man has beenassociated with elevated type-1 cytokines, and previous studies havealso shown that both TNFα and IFNγ must be present for spontaneousabortions to be induced (Chaouat et al. 1999; Tsuda et al. 2001; Clarket al. 2001b; Arck et al. 1999 and Arck et al. 1997a). Since the fgl2gene is activated by IFNγ but not by TNFα, the inventors havehypothesized that the obligatory role for the latter cytokine eitherinvolves activation of PMNL essential for abortions to be completed, ordown-regulates OX-2 expression.

[0259] It is worthy of note that in the inventors' previous work, and inthe studies described above, expression of fgl2, a thrombosis-inducingmolecule, was up-regulated on the trophoblast in response to cytokines.Cytokine-treated IRF1^(−/−) females mated to +/+ males do not abort, asthere is no up-regulation of fgl2 in the maternal decidual tissues andthe best explanation for lack of abortions is that fgl2⁺ trophoblast andfgl2⁺ maternal decidua must meet. In the regions where the two tissuesmeet, a zone of spontaneous cleavage, enough enzymatic activitypresumably occurs to cause necrosis. The inventors have also documenteda basal level of fgl2 expression in trophoblast tissue. Given theevidence that excessive anticoagulation with heparin or hirudin leads toretroplacental hemorrhage fatal to the embryo and sometimes mother, theinventors suggest that this low (basal) level of expression of themolecule fgl2 may reflect a normal homeostatic role for fgl2 inpreventing spontaneous bleeding. However, cytokine-mediated (by TNFα andIFNγ) upregulation of fgl2 is associated with increased rates ofabortion. These effects can in turn be counteracted by the combinationof both TGF-β and IL-10, both perhaps produced by trophoblast cells,which are known to inhibit cell-mediated vascular injury and clotting.There is little data to date examining the role of fgl2 prothrombinasein allograft rejection. Interestingly xenograft rejection, a process inwhich acute and subacute vascular changes are believed crucial, isreportedly less pronounced in an fgl2 knockout mouse (Levy et al,personal communication). It thus becomes extremely interesting to knowwhether fgl2 has a more general role in immunomodulation in both alloand fetal grafts.

[0260] The present data shed further light on the mechanisms by whichsome of these changes occur in both the allopregnant mouse and inallotransplant, by providing evidence for a crucial role for alteredexpression of another molecule OX2, in regulating both embryo executiontriggered by cytokine up-regulation of fgl2 prothrombinase and themodulation of renal allograft rejection. The inventors have proposedthat OX2 acts in transplantation as a co-stimulatory signal thatdeviates cytokine production away from Th1 (e.g. IL-2, IFN-γ) andtowards Th2/3 (e.g. IL-4, IL-10, TGF-β) production. Associated with thisis expansion of a γδT cell subset that mediates tolerance viasuppression. In support of such a hypothesis, the inventors have shownincreased expression of OX2 is associated with decreased rejection andaltered cytokine production; that the kinetics of expression of OX2parallels altered cytokine production and γδT cell expansion; and thatthese effects are diminished by infusion of anti-OX2 mAbs, and enhancedby infusion of the immunoadhesin OX2:Fc. The inventors have now alsodocumented that a similar correlation exists between OX2 expression andfetal loss in allopregnant mice, even when abortion rates are increasedfollowing infusion of cytokines (where our data suggests OX2 continuesto act to counter the effects of fgl2).

[0261] It is now known that OX2 functions following interaction with itsreceptor (OX2^(r)) on target cells. At least 2 groups, ourselves(Gorczynski et al. 2000 and Wright et al. 2000) have documented theexistence of OX2^(r) on macrophages, and the inventors showed optimalinhibition of graft rejection in vivo occurred with infusion of bothOX2:Fc and OX2r+ cells (Gorczynski et al. 2000). Unlike the Barclaygroup, the inventors have found that a large percentage (>80%) of ConAactivated γδT cells also express an OX2^(r), as defined by FACS withFITC-OX2:Fc. The inventors have recently confirmed this independentlyusing our mAbs to OX2r, and following cDNA sequencing of the OX2rexpressed in γδT TCR+ hybridomas (Yu et al: manuscript in preparation).The inventors have not yet studied the functional activity of OX2r cells(whether macrophages or γδT T cells) in allopregnant mice. However,extrapolating from the data shown above, it is suggested that increasedexpression of OX₂ ^(r) will correlate with successful allopregnancy, andthat triggering intracellular signaling by cross-linking OX2r on cellsby mAb (presumably in the same fashion as native cell-bound OX2 doeswhen it interacts with OX2^(r)) rescues putatively doomed embryos micefrom cytokine-induced spontaneous abortion (mediated by elevated fgl2expression).

Example 9 OX-2 Expression Rescues Putatively Doomed Embryos

[0262] The importance of CD200 (OX-2) in rescuing potentially doomedmouse embryos is illustrated by the in situ hybridization result in FIG.21. The pattern of hybridization with anti-sense probe for fgl2 (toppanels) is compared with the pattern of hybridization using anti-senseprobe for OX-2 for CBAxDBA/2 mated mice on day 8.5 of gestation. (Sensecontrols show no staining). Details of methods are provided in MolecularHuman Reproduction 2001 (Clark et al. 2001). In section 1, left, fgl2 is++and OX-2 essentially negative. In section 2, fgl2 is still ++ but inthis implantation, OX-2 hybridization can be seen in the same areas asfgl2. In section 3, fgl2 is only weakly expressed, and OX-2 is readilydemonstrable. It has been shown that the observed rate of abortions isless than expected from the % of implantations showing fgl2⁺⁺. Whenanti-OX-2 neutralizing antibody is administered, all of the fgl2⁺⁺implantation appear to be aborted. This is attributed to the demise ofimplantations showing pattern #2.

Example 10 OX-2 is Expressed in Successful Human Pregnancy

[0263] The importance of OX-2 (CD200) is rescue of potentially doomedfgl2^(hi) mouse implantations has been shown above. Increased fgl2expression is also seen in abortion of chromosomally normal humanembryos in the first trimester, but not in chromosomally abnormalembryos. To determine if OX-2 is expressed in successful pregnancies(for which first trimester tissue is not available), term placentae wereexamined. The placentae were obtained after delivery, the decidua wasscraped off, and the trophoblast cells were isolated by sequentialenzymatic digestion, as described by Guilbert et al. To removenon-trophoblast cells, anti-CD9 antibody was added, and the suspensionwas passed over an anti-immunoglobulin column, according to the methodof Guilbert et al. The adhered CD9⁺ ‘stroma’ was obtained by physicalagitation of the beads of the column. RNA was extracted in the usualmanner from a similar number of CD9⁺ and CD9⁻ cells and RT-PCR wasperformed (30 cycles) using primers rto the full-length (3 exon) OX-2gene, using primers to the 3rd exon that detects the truncated (exon2-missing) mRNA. The PAGE gel in FIG. 22 shows the molecular size ladder(lane 1), trophoblast with full length primers (lane 2) and exon 3primers (lane 3). The full length and shorter mRNA OX-2 transcripts areseen. Lane 4 (corresponding to lane 2) and lane 5 (corresponding to lane3) represent negative result obtained with CD9⁺ stromal cells.

[0264] To test for OX-2 protein expression on trophoblast, the flowcytometry protocol (summarized at the end) was used with the CD9⁻fraction. Surface expression of OX-2 was detected using PE-taggedmonoclonal antibody to human OX-2 (Cedarlane labs). Intracellularcytokeratin was stained using FITC-mAB from Dako. FIG. 23 shows resultusing antibody detecting CK5,6,8,17; similar results have been obtainedusing FITC-anti-CK18. The upper left panel shows the forward and sidescatter pattern. FITC and PE isotype controls used to set gates is shownbelow on left. Upper right panel shows cytokeratin-positive cells, themajority of which were also OX2⁺. A significant population of CK− OX2⁺⁺cells was also noted. The identity of this population has not beenestablished. On the right, the middle panel shows staining profile forOX-2 for the CK− and CK⁺ populations, and the lower panel shows thescatter. Clearly these populations have different properties. Velocitysedimentation separation indicates both populations may haveOX-2-dependent immunoregulatory activity.

Example 11 OX-2 Mab in the Prevention of Postcoital Pregnancy

[0265] The inventors have shown that infusion of an immunoadhesin,OX-2:Fc, reduces the rate of abortion in a mouse model system usinganimals with increased rates of spontaneous abortion. Antibodies to OX-2on the other hand were shown to increase the rates of spontaneousabortion, and decrease litter size in this model. This allows themanipulation of fertility using non-hormonal methods.

[0266] This example confirms the ability of OX-2 Mab to reduce fertilityin rodents.

[0267] The objection of this experiment is to determine if OX-2 Mab iscapable of preventing or reducing the fertility in rodents after coitus.

[0268] Reduction in fertility is defined as:

[0269] a) the percent of treated impregnated females delivering pups isless than that observed in the control group.

[0270] b) the average litter size of treated impregnated femalescarrying a litter to term is less than that observed in the controlgroup.

Materials and Methods

[0271] Rodents (rats/mice) were housed in individual cages with onefemale and one male/cage. Animals were inspected daily for vaginalplugs. Beginning on the day of fertilization (defined as the presence ofa vaginal plug) females were kept one to a cage, and received ipinjections of mAbs to OX2 rat anti-mouse (clone 3B6) for mice and mouseanti-rat (clone 6C2) for rats. The control group of the impregnatedfemales received injections with control rat or mouse Ig.

[0272] Records were kept of the number of impregnated females producinglitters in each group, as well as the litter size for each mother.

Animals

[0273] Mouse female, male—outbred Swiss-Webster.

[0274] Rat female, male—outbred Sprague-Dawley.

Statistical Considerations

[0275] Comparison of the percent of impregnated females carrying littersto term, and the mean litter size for successful pregnancies, were madeindependently for rats/mice using non parametric Chi-square or Fisher'sExact test where appropriate for pregnancy rates, and the Rank Sum testfor litter sizes.

Methodology Breeding

[0276] One animal of each sex is placed in a cage. The females arechecked daily for vaginal plugs. Once a vaginal plug was observed thefemales were placed one to a cage.

[0277] Beginning on the day of impregnation (defined as the day avaginal plug is observed), each female was moved to a new cage (housedalone) and begins treatment with mouse or rat OX2 antibodies byintraperitoneal injection.

Experiment No. 1

[0278] Mice: dose 100 micrograms per mouse every 3 days for 7 doses.

[0279] Rats: dose 300 micrograms per rat every 3 days for 7 doses

Experiment No.2

[0280] Mice: dose 250 micrograms per mouse every 36 hours for up to 18days

[0281] Rats: dose 500 micrograms per rat every 3 days for 7 doses

Methods of Assessment

[0282] Assessment of coitus: vaginal plug.

[0283] Assessment of pregnancy—number of pups delivered

[0284] In the second experiment the sex was also determined in each pupdelivered.

[0285] The number of animals giving birth is counted as are the numberof pups delivered.

[0286] Each female animal is housed in an individual cage until day 25after breeding. Animals not delivering by day 25 were considered not tobe pregnant and destroyed.

Study end Points

[0287] Number of animals in each group who delivered any pups.

[0288] Number of pups delivered to each animal achieving delivery atterm.

Duration of the Study

[0289] Length of time the animal were held post impregnation . . . 25days.

[0290] Methods of termination . . . pups were sacrificed after recordingthe total litter size per female. In the second study the sex of eachpup was also recorded.

[0291] Tissue to be taken . . . none.

Schedule of Evaluations

[0292] Daily inspection of animals for impregnation (vaginal plugging)

[0293] Injection of pregnant animals every 3 days in the rat experimentsand experiment 1 for the mice. The mice in experiment two were observedevery 36 hours.

[0294] Daily inspection from day 19 for evidence of birthing.

Test Article and Concomitant Treatments Test Article

[0295] Anti-OX2 mAbs were obtained from BioSpark, Canada.

[0296] Mouse anti-rat OX2: clone 6C2. Cat # SP 300X. Lot # 02

[0297] Stored at 1 mg/ml (no azide) in PBS.

[0298] Rat anti-mouse OX2: clone 3B6. Cat # SP 200X Lot # 03

[0299] Stored at 1 mg/ml, no azide, in PBS.

[0300] Control Ig was also obtained from BioSpark, Canada. Thisrepresents a preparation from pooled normal ascites (from rats or mice).

[0301] Again storage is in PBS, with no azide, at 1 mg/ml.

[0302] Normal rat Ig: Lot #01. Cat. # SP89

[0303] Normal mouse Ig: Lot # 01; Cat # SP99

[0304] Inventory and accountability records are kept by the investigator

[0305] a) The investigator kept study drugs in a locked freezer, asecure storage facility, in his laboratory at the TGH (MBRW-2-926).

Experiment 1 Mice

[0306] Control: 17/17 mice impregnated had successful pregnancies.

[0307] Mean lifter size: 7

[0308]3B6 injected: 17/18 mice impregnated had successful pregnancies.

[0309] Mean lifter size: 6

[0310] See Table 8

[0311] Statistics: Mice: 17 control and 18 treated

[0312] The proportion of mice pregnant was not significantly differentby Fisher's Exact test; the litter size in each group, and the averagenumber of pups per group were not significantly different by the RankSum test.

Rats

[0313] Control: 11/12 rats impregnated had successful pregnancies.

[0314] Mean litter size: 9

[0315] 6C2 injected: 5/8 rats impregnated had successful pregnancies.

[0316] Mean litter size for the group was 5 but for the 5 ratsdelivering pups the mean litter size was 8

[0317] See Table 10

[0318] P<0.05 for successful pregnancies.

[0319] Statistics: Rats 12 control and 8 treated

[0320] The proportion of animals pregnant in the treated group was notsignificantly reduced (P=0.153, Fisher's Exact test). Litter size inthose delivering was reduced (P<0.018, Rank Sum test), and the totalnumber of pups generated by the immune treated group was reduced(P<0.006, Rank Sum test)

Experiment 2 Mice

[0321] Control: 8/8 mice impregnated had successful pregnancies.

[0322] Mean litter size: 6

[0323] 3B6 injected: 3/8 mice impregnated had successful pregnancies.

[0324] Mean litter size for the group was 2 but for the 3 ratsdelivering pups the mean litter size was 5

[0325] See Table 9

[0326] Statistics: Mice 8 control and 8 treated

[0327] The proportion of rats pregnant was significantly reduced bytreatment (P=0.0128, Fisher's Exact test), and the number of pupsoverall was reduced (P<0.00021, Rank Sum test). The average litter sizefor animals successfully delivering was also reduced (P<0.024, Rank Sumtest), but the number of litters was small.

Rats

[0328] Control: 6/6 rats impregnated had successful pregnancies.

[0329] Mean litter size: 10

[0330] 6C2 injected: 2/6 rats impregnated had successful pregnancies.

[0331] Mean litter size for the group was 2.5 but for the two ratsdelivering pups the mean litter size was 7.5

[0332] See Table 11

[0333] Statistics: Rats 6 control 6 treated

[0334] The proportion of animals that delivered litters wassignificantly reduced by treatment (P=0.03, Fisher's Exact test), as wasthe total number of pups produced by the treated group (P<0.002, RankSum test). Litter size was also slightly reduced in those delivering(P<0.03, Rank Sum test), but the number of litters was small.

Conclusions

[0335] The administration of OX-2 antibodies to rats and mice followingimpregnations has been shown to reduce the number of animals deliveringpups. The average liter size of antibody treated animals that diddeliver was not different from the control animals.

[0336] While the present invention has been described with reference towhat are presently considered to be the preferred examples, it is to beunderstood that the invention is not limited to the disclosed examples.To the contrary, the invention is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

[0337] All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety. TABLE 1 Summary of sequences and clones detected incDNA library from pv immunized mice Match category Number of clonesrepresented (%) Known mouse genes 30 (45) Non-mouse genes (rat/human) 1014 (21) No data base match 22 (34)

[0338] TABLE 2 Cytokine production from cells of mice receiving pvimmunization and anti-rat OX-2 Cytokine levels in culturesupernatants^(b) Mabs given to recipients^(a) IL-2 IFNγ IL-4 IL-10 No pvimmunization (CsA only) None 750 ± 125  85 ± 18 29 ± 8 130 ± 40 +anti-rat OX-2 890 ± 160  93 ± 19  30 ± 10 120 ± 35  +anti-mouse CD28415 ± 88*  57 ± 9*  105 ± 22* 275 ± 55* +anti-mouse CTLA4  505 ± 125* 65± 8  95 ± 20* 190 ± 45  +anti-B7-1 340 ± 65*  35 ± 7*  120 ± 21* 285 ±60* +anti-B7-2 495 ± 90* 64 ± 7  90 ± 20* 185 ± 45  PV Immunization +CsA None 190 ± 55  25 ± 8 107 ± 21 780 ± 150 +anti-rat OX-2  730 ± 140* 60 ± 16*  33 ± 10* 220 ± 40* +anti-mouse CD28 145 ± 38  20 ± 9 145 ± 341140 ± 245  +anti-mouse CTLA4 85 ± 25 15 ± 6 125 ± 31 960 ± 220+anti-B7-1 110 ± 30  20 ± 6 144 ± 28 885 ± 180 +anti-B7-2 75 ± 20 14 ± 5150 ± 30 1230 ± 245 

[0339] TABLE 3 FACS staining of PBL and spleen adherent cells indifferent species, using anti-OX-2 Mabs Donor^(b) Percent stainedcells^(c) SPECIES^(a) Treatment Mab PBL Spleen Human NONE H4B4 1.5 ± 0.34.8 ± 1.7 H4A9A2 1.5 ± 0.4 6.1 ± 2.0 H4A9C7 1.3 ± 0.4 4.3 ± 1.7 MouseNONE M3B5 1.9 ± 0.4 6.7 ± 2.1 M3B6 1.7 ± 0.4 5.2 ± 1.6 M2C8 1.4 ± 0.44.2 ± 1.4 Mouse PV immune M3B5 5.9 ± 1.5  20 ± 4.1 M3B6 5.2 ± 1.4  17 ±3.6 M2C8 4.7 ± 1.4  15 ± 3.3 Rat NONE RC6A3 1.3 ± 0.3 5.3 ± 1.6 RC6C21.5 ± 0.4 6.5 ± 1.7 RC6D1 1.9 ± 0.6 6.8 ± 1.5 Rat PV immune RC6A3 4.8 ±1.3  16 ± 4.2 RC6C2 4.9 ± 1.6  18 ± 3.9 RC6D1 5.3 ± 1.7  20 ± 4.5

[0340] TABLE 4 Type-1 cytokine production in MLR cultures is increasedby anti-OX-2 Mabs Cytokine levels in culture supernatants^(b) ELISAassays (murine only) Bioassay (CTTL-2) Mabs in culture^(a) IL-2 IFNγIL-4 IL-10 IL-2 IL-6 MOUSE MLR None 350 ± 55  35 ± 18 345 ± 63  340 ±50  480 ± 160  365 ± 74 M3B5 890 ± 160* 115 ± 29* 130 ± 10* 168 ± 42*820 ± 200* 265 ± 46 M3B6 915 ± 155* 117 ± 25* 135 ± 32* 135 ± 38* 850 ±175* 303 ± 55 M2C8 855 ± 155* 105 ± 28* 120 ± 32* 140 ± 37* 830 ± 165*279 ± 61 control lg 370 ± 75  36 ± 11 330 ± 55  310 ± 45  335 ± 60  349± 59 None** 710 ± 145  108 ± 23  110 ± 21  105 ± 23  690 ± 155  285 ± 54RAT MLR None 490 ± 145  360 ± 57 RC6A3 690 ± 155* 295 ± 55 RC6C2 845 ±180* 345 ± 68 RC6D1 830 ± 160* 370 ± 57 Control lg 475 ± 160  356 ± 58HUMAN MLR None 395 ± 85  295 ± 45 H4B4 570 ± 125* 315 ± 50 H4A9A2 630 ±145* 320 ± 48 H4A9C7 625 ± 140* 345 ± 56 Control lg 360 ± 120  320 ± 50#set up in triplicate for each Mab. Mouse responder spleen cells werefrom mice treated 4 days earlier by portal vein infusion of C57BL/6 bonemarrow cells, except for data shown as (None**) where responder cellswere from non-injected C3H mice. Mab was added as a 30% superntatantconcentration. Supernatants were harvested for cytokine assays at 60hrs. #or FACS) gave cytokine data indistinguishable from culturesincubated in the absence of Mab. p < 0.05, compared with cultureswithout Mabs.

[0341] TABLE 5 OX-2:FC Immunoadhesin Inhibits Mixed Leukocyte Reactionin vitro Added supernatant^(a) Percent lysis ⁵¹Cr targets^(b) Cytokinesin culture (pg/ml)^(c) (50:1, effector:target) IL-2 IL-4 NONE (control)31 ± 4.0 1005 ± 185 60 ± 20 Control CHO 33 ± 4.3  810 ± 190 45 ± 20(vector transduced) CHO transduced with 4.2 ± 2.1  175 ± 45 245 ± 55 OX-2:Fc

[0342] TABLE 6 Inhibition of skin graft rejection by OX-2:Fc Treatmentof mice Rejection of skin grafts (mean + SD) in days NIL 12 + 3.8OX-2:Fc 19 + 4.2

[0343] TABLE 7 OX-2:Fc infused into mice receiving skin allograftsreverses polarization in cytokine production Treatment of mice Cytokinesin culture supernatant at 48 hrs (pg/ml) IL-2 IL-4 NIL 1250 + 160 80 +20 OX-2:Fc 350 + 85 245 + 50 

[0344] TABLE 8 Mice Experiment Mouse type: Swiss-Webster-Outbred TestArticle: Clone 3B6rat - anti-mouse OX-2 Dose 100 pg IP Q 3 days Placebo:Mouse lgg Controls Mouse Date Delivery Number Plugged Date Litter size 1 Feb-28-01 Mar-21 8 Controls % Pregnant 100  2 Feb-29 Mar-21 6  3Feb-29 Mar-21 8  4 Mar-01 Mar-22 6  5 Mar-01 Mar-22 6  6 Mar-03 Mar-24 8 7 Mar-03 Mar-24 4  8 Mar-05 Mar-26 5  9 Mar-05 Mar-26 8 10 Mar-06Mar-27 8 11 Mar-06 Mar-27 6 12 Mar-06 Mar-28 8 13 Mar-08 Mar-29 8 14Mar-08 Mar-29 6 15 Mar-08 Mar-30 4 16 Mar-08 Mar-30 8 17 Mar-11 Mar-31 6Treated  1 Feb-28 Mar-20 7  2 Feb-29 Mar-21 8  3 Feb-29 Mar-21 8Treated: % Pregnant  94  4 Feb-29 Mar-21 4  5 Mar-01 Mar-22 5  6 Mar-03Mar-22 8  7 Mar-03 Mar-23 8  8 Mar-05 Mar-25 6  9 Mar-05 Mar-25 6 10Mar-06 Mar-25 5 11 Mar-06 Mar-26 4 12 Mar-06 Mar-27 4 13 Mar-06 Mar-27 814 Mar-08 Nil 0 15 Mar-08 Mar-29 6 16 Mar-08 Mar-29 7 17 Mar-08 Mar-29 818 Mar-11 Mar-31 7

[0345] TABLE 9 Mice Experiment Mouse type: Swiss-Webster-Outbred TestArticle: Clone 3B6rat - anti-mouse OX-2 Dose 250 micrograms IP every 36hours Placebo: Mouse lgg Controls Mouse Date Delivery Litter Size LitterSize Litter Size Number Plugged Date Males Females Total 1 Apr-01 Apr-214 2 6 2 Apr-01 Apr-22 4 3 7 3 Apr-03 Apr-25 3 3 6 4 Apr-05 Apr-26 2 4 65 Apr-05 Apr-27 5 3 8 6 Apr-08 Apr-29 4 4 8 7 Apr-09 Apr-30 3 3 6 8Apr-10 Apr-30 2 4 6 Treated Mouse Date Delivery Litter Size Litter SizeLitter Size Number Plugged Date Males Females Total 1 Apr-01 Nil 0 0 0 2Apr-02 Nil 0 0 0 3 Apr-04 Apr-25 4 2 6 4 Apr-05 Nil 0 0 0 5 Apr-07Apr-29 1 3 4 6 Apr-09 Apr-30 4 2 6 7 Apr-10 Nil 0 0 0 8 Apr-13 Nil 0 0 0Controls % Pregnant 100 Treated % Pregnant  38

[0346] TABLE 10 Rat Experiment Rat type: Sprague-Dawley-Outbred TestArticle: Clone 6C2 Mouse - anti-rat OX-2 Dose 300 micrograms IP Q 3 daysPlacebo: Rat lgg Controls RAt Date Delivery Number Plugged Date Littersize 1 Feb-29 Mar-21 11 2 Feb-29 Mar-22 13 Controls % Pregnant 92 3Mar-01 Mar-24 10 4 Mar-01 Mar-24 11 5 Mar-03 Mar-25 12 6 Mar-03 Mar-25 9 7 Mar-05 Nil  0 8 Mar-05 Mar-27  9 9 Mar-06 Mar-28  8 10  Mar-07Mar-28  9 11  Mar-07 Mar-29 10 12  Mar-09 Mar-31  8 Treated Rat DateDelivery Number Plugged Date Litter size 1 Feb-29 Mar-21 8 Treated: %Pregnant 63 2 Mar-01 Mar-23 9 3 Mar-01 Nil 0 4 Mar-01 Mar-24 9 5 Mar-03Nil 0 6 Mar-05 Nil 0 7 Mar-06 Mar-28 8 8 Mar-07 Mar-29 6

[0347] TABLE 11 Rat Experiment Rat type: Sprague-Dawley-Outbred TestArticle: Clone 6C2 Mouse - anti-rat OX-2 Dose 500 micrograms IP Q 3 daysPlacebo: Rat lgg Controls RAt Date Delivery Litter Size Litter SizeLitter Size Number Plugged Date Males Females Total 1 Apr-04 Apr-25 6 410 2 Apr-04 Apr-27 6 5 11 3 Apr-08 Apr-29 4 6 10 4 Apr-09 Apr-30 3 7 105 Apr-13 May-02 4 5  9 6 Apr-14 May-03 6 5 11 Controls % Pregnant 100Treated Rat Date Delivery Litter Size Litter Size Litter Size NumberPlugged Date Males Females Total 1 Apr-05 Nil 0 0 0 2 Apr-06 Nil 0 0 0 3Apr-09 Apr-30 3 4 7 4 Apr-12 Nil 0 0 0 5 Apr-14 Nil 0 0 0 6 Apr-16May-06 4 4 8 Treated: % Pregnant  33

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1 22 1 2791 DNA Mus musculus 1 actatagggc acgcgtggtc gacggcccgggctggtactg agaaggaata ggatgcagtc 60 agagggaagg gacttgagga agacctttggtttagactct ctccacatgt ctgtctgtgg 120 gtctctgaac cagattttat ctgttgctgcctctctgatg acagctggtc aaggccccaa 180 tctattagta tagcagaatg tcattaagaatcattctttt cttccttcca ttttttcttc 240 ttcttctact ccctccccct ttctctctctctctttcttt ctttctttct ttctttttct 300 ttctttcttt ctctctctcc ctctttctctctctccctct ctctctttct ttctttcttt 360 ctttctcttt ttctttctgt ctttctctctctttccttct ttccttcccc agctgtgttt 420 ggttctaccc taggactctg ggctttccattatctggttc ttgaccatcc aggcaatata 480 ggtacaagct ctctcttata gggtgggcctcaagttaaac taaacattgg ttggtcactc 540 cctcacgttt tctactaaaa tctcataggcaggacatatt gtgggtagag gatttagagg 600 caggtttagt gtccaggttt gtcttttcatggtctgtaga ataccttctc acaccagaga 660 gactagagtc tagagtccaa acctcagctctagcctctct atgttcagtg agctgaatga 720 aagttgacct cagcaatggg tcccactgtcaggttttaga gggtgacctt cagttgtagg 780 tcccaagtct ctctctcctc tctctccctttctcgatctc tctctctctc tctctctctc 840 tctctctctc tctctctctc tctctctctctctctctgct ttatacttgt gattgaagat 900 gtgatctctc tggcagcctg gtaccatgcctcctggtcac ttagagactc tcctcctgta 960 gctataagcc caacaaatct ttttccacaggtttctactc tagtacagaa acagaaatgt 1020 caccaatata gtcaatcgtt tctgtaaagctttcatcaag gaaaacctca gttccagggc 1080 ttcctgtgac tcatttgatc tgtcccttgattctcatctg ttttaaggaa tactgcggga 1140 caatctgatt agcagaaaga aagtgcttttgggttttcag gaagtgtgtt cacaggtagc 1200 tctgagccct taggacttct aaagctctagatgaggtacc tggtaaccac acacacacac 1260 acacacacac acacacacac acacgcactggcctttaata taacaaatca taaaataaag 1320 tttttctttt tttttcccca gggtgtctgtatgaatctcc ttaccttctt ccccctacac 1380 acacacacac acacacacac acacacacacacactattgt tctgttctcc gagtttacct 1440 tttgctgtac agaaccacag gatgcaccgggtttctgact caaattactg tccactcaag 1500 ttagttccca ctccgatttt tctgtatggactacgtcacc ctatactgcc atttggcacg 1560 ggagagaggc cagtgatggg aatgcagacgaaacatgcat acacatgtaa aataagataa 1620 ataaatctaa aatgaaaaaa aatatagagtgattctttca catttttgct atattactct 1680 aaaaggcgag aacctggcgg gggcgggggcaggggctagg gacgaggttg tagagggcgt 1740 ggttggttgg tcgtctcttc ctccacactagaggagctgt agagtctgcc tgtgcggtgg 1800 agggggctct ctctacggcg aatagtagtgtccctgctca caggtgttgc ggagatatcc 1860 tccatcgtgg aagagctcag accccgagaagctggtgtct agctgcggcc ccgagcaagg 1920 atgggcagtc tggtgagtgg aatctgagatgcgaaggagg gcggaatggg cgatctggag 1980 ccgcggctct cagaagccag tggagcctgcgagaaaagca aggaagctgt tctttggaga 2040 agtggtatcc ggggctcgga gctctgtaaggaggcaccgg ccggagaaag cccggggaac 2100 gcgtgtatct agggtgggcg gctttgctccttgctgcgat tccattgcga aaacacggcc 2160 tgagctccat ggctcccaga aggggaggagtagctctttg cgtcccctat gttggtcctt 2220 aacctgcagc aggggtgtag cctagtaatctcgcttgctc tctttctcac cccctctctt 2280 gctgcatttc tgctccttgc ctagaaaaccatgaagcatc tagcagtact gcagcgagca 2340 agccacagct tagtggtctt gttaaatgccaaggtattta gaggagaggc cgacattttg 2400 agtctttggt actgtttaca aggcagaaaattttaaaagg aagggtggtc atacgcctta 2460 ttctttatac acacggaatt ggtagaattgaatgcgaatc taaacgcaat taaaccccag 2520 gtaccacttt tcatcaggct gacaaagaccgacttgtgtt acctttccta acaaagagga 2580 atgtggatct gtcagctaga tgctcttagtgttcaaacaa ggaattgctt tctgttttac 2640 aaagaatcgg agagagaggt tcttttttttctctccaagt ctctgtggct gcaatgaaat 2700 aaggtacaaa atcagaccta gaaagaataggggaatgggg ctatgcacct agcagaccag 2760 cccgggccgt cgaccacgcg tgccctatag t2791 2 278 PRT Mus musculus 2 Met Gly Ser Leu Val Phe Arg Arg Pro PheCys His Leu Ser Thr Tyr 1 5 10 15 Ser Leu Ile Trp Gly Met Ala Ala ValAla Leu Ser Thr Ala Gln Val 20 25 30 Glu Val Val Thr Gln Asp Glu Arg LysAla Leu His Thr Thr Ala Ser 35 40 45 Leu Arg Cys Ser Leu Lys Thr Ser GlnGlu Pro Leu Ile Val Thr Trp 50 55 60 Gln Lys Lys Lys Ala Val Ser Pro GluAsn Met Val Thr Tyr Ser Lys 65 70 75 80 Thr His Gly Val Val Ile Gln ProAla Tyr Lys Asp Arg Ile Asn Val 85 90 95 Thr Glu Leu Gly Leu Trp Asn SerSer Ile Thr Phe Trp Asn Thr Thr 100 105 110 Leu Glu Asp Glu Gly Cys TyrMet Cys Leu Phe Asn Thr Phe Gly Ser 115 120 125 Gln Lys Val Ser Gly ThrAla Cys Leu Thr Leu Tyr Val Gln Pro Ile 130 135 140 Val His Leu His TyrAsn Tyr Phe Glu Asp His Leu Asn Ile Thr Cys 145 150 155 160 Ser Ala ThrAla Arg Pro Ala Pro Ala Ile Ser Trp Lys Gly Thr Gly 165 170 175 Thr GlyIle Glu Asn Ser Thr Glu Ser His Phe His Ser Asn Gly Thr 180 185 190 ThrSer Val Thr Ser Ile Leu Arg Val Lys Asp Pro Lys Thr Gln Val 195 200 205Gly Lys Glu Val Ile Cys Gln Val Leu Tyr Leu Gly Asn Val Ile Asp 210 215220 Tyr Lys Gln Ser Leu Asp Lys Gly Phe Trp Phe Ser Val Pro Leu Leu 225230 235 240 Leu Ser Ile Val Ser Leu Val Ile Leu Leu Val Leu Ile Ser IleLeu 245 250 255 Leu Tyr Trp Lys Arg His Arg Asn Gln Glu Arg Gly Glu SerSer Gln 260 265 270 Gly Met Gln Arg Met Lys 275 3 14 DNA ArtificialSequence Primer 3 ttttgtacaa gctt 14 4 44 DNA Artificial SequenceAdapter 1 4 ctaatacgac tcactatagg gctcgagcgg ccgcccgggc aggt 44 5 43 DNAArtificial Sequence Adapter 2 5 tgtagcgtga agacgacaga aagggcgtggtgcggagggc ggt 43 6 22 DNA Artificial Sequence Primer 1 6 ctaatacgactcactatagg gc 22 7 22 DNA Artificial Sequence Nested Primer 1 7tcgagcggcc gcccgggcag gt 22 8 21 DNA Artificial Sequence Primer 2 8tgtagcgtga agacgacaga a 21 9 22 DNA Artificial Sequence Nested Primer 29 agggcgtggt gcggagggcg gt 22 10 25 DNA Artificial Sequence GADPH Sense10 tgatgacatc aagaaggtgg tgaag 25 11 23 DNA Artificial Sequence GADPHAntisense 11 tccttggagg ccatgtaggc cat 23 12 20 DNA Artificial SequenceB7-1 Sense 12 ccttgccgtt acaactctcc 20 13 20 DNA Artificial SequenceB7-1 Antisense 13 cggaagcaaa gcaggtaatc 20 14 20 DNA Artificial SequenceB7-2 Sense 14 tctcagatgc tgtttccgtg 20 15 20 DNA Artificial SequenceB7-2 Antisense 15 ggttcactga agttggcgat 20 16 20 DNA Artificial SequenceOX-2 Sense 16 gtggaagtgg tgacccagga 20 17 20 DNA Artificial SequenceOX-2 Antisense 17 atagagagta aggcaagctg 20 18 825 DNA Homo sapiens 18gtgatcagga tgcccttctc tcatctctcc tcctacagcc tggtttgggt catggcagca 60gtggtgctgt gcacagcaca agtgcaagtg gtgacccagg atgaaagaga gcagctgtac 120acacctgctt ccttaaaatg ctctctgcaa aatgcccagg aagccctcat tgtgacatgg 180cagaaaaaga aagctgtaag cccagaaaac atggtcacct tcagcgagaa ccatggggtg 240gtgatccagc ctgcctataa ggacaagata aacattaccc agctgggact ccaaaactca 300accatcacct tctggaatat caccctggag gatgaagggt gttacatgtg tctcttcaat 360acctttggtt ttgggaagat ctcaggaacg gcctgcctca ccgtctatgt acagcccata 420gtatcccttc actacaaatt ctctgaagac cacctaaata tcacttgctc tgccactgcc 480cgcccagccc ccatggtctt ctggaaggtc cctcggtcag ggattgaaaa tagtacagtg 540actctgtctc acccaaatgg gaccacgtct gttaccagca tcctccatat caaagaccct 600aagaatcagg tggggaagga ggtgatctgc caggtgctgc acctggggac tgtgaccgac 660tttaagcaaa ccgtcaacaa aggatattgg ttttcagttc cgctattgct aagcattgtt 720tccctggtaa ttcttctcat cctaatctca atcttactgt actggaaacg tcaccggaat 780caggaccgag gtgaattgtc acagggagtt caaaaaatga cataa 825 19 274 PRT Homosapiens 19 Val Ile Arg Met Pro Phe Ser His Leu Ser Thr Tyr Ser Leu ValTrp 1 5 10 15 Val Met Ala Ala Val Val Leu Cys Thr Ala Gln Val Gln ValVal Thr 20 25 30 Gln Asp Glu Arg Glu Gln Leu Tyr Thr Thr Ala Ser Leu LysCys Ser 35 40 45 Leu Gln Asn Ala Gln Glu Ala Leu Ile Val Thr Trp Gln LysLys Lys 50 55 60 Ala Val Ser Pro Glu Asn Met Val Thr Phe Ser Glu Asn HisGly Val 65 70 75 80 Val Ile Gln Pro Ala Tyr Lys Asp Lys Ile Asn Ile ThrGln Leu Gly 85 90 95 Leu Gln Asn Ser Thr Ile Thr Phe Trp Asn Ile Thr LeuGlu Asp Glu 100 105 110 Gly Cys Tyr Met Cys Leu Phe Asn Thr Phe Gly PheGly Lys Ile Ser 115 120 125 Gly Thr Ala Cys Leu Thr Val Tyr Val Gln ProIle Val Ser Leu His 130 135 140 Tyr Lys Phe Ser Glu Asp His Leu Asn IleThr Cys Ser Ala Thr Ala 145 150 155 160 Arg Pro Ala Pro Met Val Phe TrpLys Val Pro Arg Ser Gly Ile Glu 165 170 175 Asn Ser Thr Val Thr Leu SerHis Pro Asn Gly Thr Thr Ser Val Thr 180 185 190 Ser Ile Leu His Ile LysAsp Pro Lys Asn Gln Val Gly Lys Glu Val 195 200 205 Ile Cys Gln Val LeuHis Leu Gly Thr Val Thr Asp Phe Lys Gln Thr 210 215 220 Val Asn Lys GlyTyr Trp Phe Ser Val Pro Leu Leu Leu Ser Ile Val 225 230 235 240 Ser LeuVal Ile Leu Leu Val Leu Ile Ser Ile Leu Leu Tyr Trp Lys 245 250 255 ArgHis Arg Asn Gln Asp Arg Gly Glu Leu Ser Gln Gly Val Gln Lys 260 265 270Met Thr 20 837 DNA Rattus norvegicus 20 atgggcagtc cggtattcag gagacctttctgccatctgt ccacctacag cctgctctgg 60 gccatagcag cagtagcgct gagcacagctcaagtggaag tggtgaccca ggatgaaaga 120 aagctgctgc acacaactgc atccttacgctgttctctaa aaacaaccca ggaacccttg 180 attgtgacat ggcagaaaaa gaaagccgtaggcccagaaa acatggtcac ttacagcaaa 240 gcccatgggg ttgtcattca gcccacctacaaagacagga taaacatcac tgagctggga 300 ctcttgaaca caagcatcac cttctggaacacaaccctgg atgatgaggg ttgctacatg 360 tgtctcttca acatgtttgg atctgggaaggtctctggga cagcttgcct tactctctat 420 gtacagccca tagtacacct tcactacaactattttgaag accacctaaa catcacgtgc 480 tctgcaactg cccgcccagc ccctgccatctcctggaagg gcactgggtc aggaattgag 540 aatagtactg agagtcactc ccattcaaatgggactacat ctgtcaccag catcctccgg 600 gtcaaagacc ccaaaactca ggttggaaaggaagtgatct gccaggtttt atacttgggg 660 aatgtgattg actacaagca gagtctggacaaaggatttt ggttttcagt cccactgctg 720 ctgagcattg tttctctggt aattcttctggtcttgatct ccatcttatt atactggaaa 780 cggcaccgaa atcaggagcg gggtgagtcatcacagggga tgcaaagaat gaaataa 837 21 278 PRT Rattus norvegicus 21 MetGly Ser Pro Val Phe Arg Arg Pro Phe Cys His Leu Ser Thr Tyr 1 5 10 15Ser Leu Leu Trp Ala Ile Ala Ala Val Ala Leu Ser Thr Ala Gln Val 20 25 30Glu Val Val Thr Gln Asp Glu Arg Lys Leu Leu His Thr Thr Ala Ser 35 40 45Leu Arg Cys Ser Leu Lys Thr Thr Gln Glu Pro Leu Ile Val Thr Trp 50 55 60Gln Lys Lys Lys Ala Val Gly Pro Glu Asn Met Val Thr Tyr Ser Lys 65 70 7580 Ala His Gly Val Val Ile Gln Pro Thr Tyr Lys Asp Arg Ile Asn Ile 85 9095 Thr Glu Leu Gly Leu Leu Asn Thr Ser Ile Thr Phe Trp Asn Thr Thr 100105 110 Leu Asp Asp Gly Gly Cys Tyr Met Cys Leu Phe Asn Met Phe Gly Ser115 120 125 Gly Lys Val Ser Gly Thr Ala Cys Leu Thr Leu Tyr Val Gln ProIle 130 135 140 Val His Leu His Tyr Asn Tyr Phe Glu His His Leu Asn IleThr Cys 145 150 155 160 Ser Ala Thr Ala Arg Pro Ala Pro Ala Ile Ser TrpLys Gly Thr Gly 165 170 175 Ser Gly Ile Glu Asn Ser Thr Glu Ser His SerHis Ser Asn Gly Thr 180 185 190 Thr Ser Val Thr Ser Ile Leu Arg Val LysAsp Pro Lys Thr Gln Val 195 200 205 Gly Lys Glu Val Ile Cys Gln Val LeuTyr Leu Gly Asn Val Ile Asp 210 215 220 Tyr Lys Gln Ser Leu Asp Lys GlyPhe Trp Phe Ser Val Pro Leu Leu 225 230 235 240 Leu Ser Ile Val Ser LeuVal Ile Leu Leu Val Leu Ile Ser Ile Leu 245 250 255 Leu Tyr Trp Lys ArgHis Arg Asn Gln Glu Arg Gly Glu Ser Ser Gln 260 265 270 Gly Met Gln ArgMet Lys 275 22 837 DNA Mus musculus 22 atgggcagtc tggtattcag gagacctttctgccatctct ccacctacag cctgatttgg 60 ggcatagcag cagtagcgct gagcacagctcaagtggaag tggtgaccca ggatgaaaga 120 aaggcgctgc acacaactgc atccttacgatgttctctaa aaacatccca ggaacccttg 180 attgtgacat ggcagaaaaa gaaagccgtgagcccagaaa acatggtcac ctacagcaaa 240 acccatgggg ttgtaatcca gcctgcctacaaagacagga taaatgtcac agagctggga 300 ctctggaact caagcatcac cttctggaacacacacattg gagatggagg ctgctacatg 360 tgtctcttca acacgtttgg ttctcagaaggtctcaggaa cagcttgcct tactctctat 420 gtacagccca tagtacacct tcactacaactattttgaac accacctaaa catcacttgc 480 tctgcgactg cccgtccagc ccctgccatcacctggaagg gtactgggac aggaattgag 540 aatagtaccg agagtcactt ccattcaaatgggactacat ctgtcaccag catcctccgg 600 gtcaaagacc ccaaaactca agttgggaaggaagtgatct gccaggtttt atacctgggg 660 aatgtgattg actacaagca gagtctggacaaaggatttt ggttttcagt tccactgttg 720 ctaagcattg tttctctggt aattcttctgatcttgatct ccatcttact atactggaaa 780 cgtcaccgaa atcaggagcg gggtgaatcatcacagggga tgcaaagaat gaaataa 837

We claim:
 1. A method of preventing, inhibiting or reducing fetal losscomprising administering an effective amount of an OX-2 protein orfragment thereof, or a nucleic acid molecule encoding an OX-2 protein orfragment thereof to an animal in need thereof.
 2. A method according toclaim 1 wherein the OX-2 protein is a human OX-2 protein or a fragmentthereof.
 3. A method according to claim 1 wherein the OX-2 protein is asoluble fusion protein.
 4. A method according to claim 3 wherein thesoluble fusion protein comprises an OX-2 protein or fragment thereoflinked to an immunoglobulin Fc region.
 5. A method according to claim 4wherein the OX-2 fragment comprises an extracellular domain of an OX-2protein.
 6. A method of inducing fetal loss comprising administering aneffective amount of an agent that inhibits an OX-2 protein to an animalin need thereof.
 7. A method according to claim 6 wherein the agent is amolecule that binds the OX-2 protein.
 8. A method according to claim 7wherein the molecule is an antibody.
 9. A method according to claim 6wherein the agent is an antisense oligonucleotide that is complimentaryto a nucleic acid sequence from an OX-2 gene.
 10. A pharmaceuticalcomposition for use in preventing, inhibiting or reducing fetal losscomprising an OX-2 protein in admixture suitable diluent or carrier. 11.A pharmaceutical composition according to claim 10 wherein the OX-2protein is a soluble fusion protein.
 12. A pharmaceutical compositionaccording to claim 10 wherein the OX-2 protein is a human OX-2 proteinor a fragment thereof.
 13. A pharmaceutical composition according toclaim 11 wherein the soluble fusion protein comprises an OX-2 protein orfragment thereof linked to an immunoglobulin Fc region.
 14. Apharmaceutical composition according to claim 13 wherein the OX-2fragment comprises an extracellular domain of an OX-2 protein.
 15. Apharmaceutical composition for use in inducing immune suppressioncomprising an effective amount of an agent that inhibits OX-2 inadmixture with a suitable diluent or carrier.
 16. A compositionaccording to claim 15 wherein the agent is a molecule that binds theOX-2 protein.
 17. A composition according to claim 16 wherein themolecule is an antibody.
 18. A composition according to claim 14 whereinthe agent is an antisense oligonucleotide that is complimentary to anucleic acid sequence from an OX-2 gene.